Frontiers in Endocrinology

Published by Frontiers
Online ISSN: 1664-2392
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Airway epithelial NF-kB activation is observed in asthmatic subjects and is a cause of airway inflammation in mouse models of allergic asthma. Combination therapy with inhaled short-acting b2-agonists and corticosteroids significantly improves lung function and reduces inflammation in asthmatic subjects. Corticosteroids operate through a number of mechanisms to potently inhibit NF-kB activity. Since b-agonists can induce expression of 11b-HSD1, which converts inactive 11-keto corticosteroids into active 11-hydroxy corticosteroids, thereby potentiating the effects of endogenous glucocorticoids, we examined whether this mechanism is involved in the inhibition of NF-kB activation induced by the b-agonist albuterol in airway epithelial cells. Treatment of transformed murine Club cells (MTCC) with (R)-albuterol (levalbuterol), but not with (S)- or a mixture of (R+S)- (racemic) albuterol, augmented mRNA expression of 11b-HSD1. MTCC were stably transfected with luciferase (luc) reporter constructs under transcriptional regulation by NF-kB (NF-kB/luc) or glucocorticoid response element (GRE/luc) consensus motifs. Stimulation of NF-kB/luc MTCC with lipopolysaccharide (LPS) or tumor necrosis factor-α (TNFα) induced luciferase activity, which was inhibited by pretreatment with (R)-, but not (S)- or racemic albuterol. Furthermore, pretreatment of GRE/luc MTCC with (R)-albuterol augmented 11-keto corticosteroid (cortisone) induced luciferase activity, which was diminished by the 11β-HSD inhibitor glycyrrhetinic acid (18β-GA). LPS- and TNFα-induced NF-kB/luc activity was diminished in MTCC cells treated with a combination of cortisone and (R)-albuterol, an effect that was inhibited by 18β-GA. Finally, pretreatment of MTCC cells with the combination of cortisone and (R)-albuterol diminished LPS- and TNFα-induced pro-inflammatory cytokine production. These results demonstrate that levalbuterol augments conversion of inactive 11-keto corticosteroids into the active 11-hydroxy form.
 
| Results of microsatellite analysis of markers on chromosome 11p amplified from leukocyte DNA of the probands and their parents and from the pancreatic DNA from the probands. Red text represents markers which are informative for
| A region of chromosome 11 has been expanded to demonstrate the area of UPD in relation to the location of the imprinted locus at 11p15.5 and the ABCC8 and KCNJ11 genes. IC denotes the imprinted control regions 1 and 2. Red text represents markers which are informative for paternal UPD, green text represents markers which show no evidence of UPD, and blue texts represents uninformative markers. Directly
Results of microsatellite analysis of markers on chromosome 11p amplified from leukocyte DNA of the probands and their parents and from the pancreatic DNA from the probands. Red text represents markers which are informative for paternal UPD, green text represents markers which show no evidence of UPD, and blue texts represents markers which are uninformative. The distance between the marker and the telomere at 11p is provided.
A region of chromosome 11 has been expanded to demonstrate the area of UPD in relation to the location of the imprinted locus at 11p15.5 and the ABCC8 and KCNJ11 genes. IC denotes the imprinted control regions 1 and 2. Red text represents markers which are informative for paternal UPD, green text represents markers which show no evidence of UPD, and blue texts represents uninformative markers. Directly below are electropherograms that demonstrate the results of microsatellite analysis for one of the markers (D11S1318 and D11S1397) which are informative for UPD in pancreatic tissue for each of the probands. The x-axis indicates the size of the product (base pairs) and the y-axis denotes the product quantity (arbitrary units). In each example mosaic paternal UPD is illustrated by a larger peak for the paternal allele compared to the maternal.
Background: Loss of function mutations in the genes encoding the pancreatic β-cell ATP-sensitive potassium (KATP) channel are identified in approximately 80% of patients with diazoxide unresponsive hyperinsulinemic hypoglycemia (HH). For a small number of patients HH can occur as part of a multisystem disease such as Beckwith-Wiedemann syndrome (BWS). In approximately 20% of patients, BWS results from chromosome 11 paternal uniparental disomy (UPD), which causes dysregulation of imprinted growth regulation genes at 11p15.5. There is a considerable range in the clinical features and phenotypic severity associated with BWS which is likely to be due to somatic mosaicism. The cause of HH in these patients is not known. Research design and methods: We undertook microsatellite analysis of 12 markers spanning chromosome 11p in two patients with severe HH and diffuse disease requiring a pancreatectomy. In both patients mutations in the K(ATP) channel genes had not been identified. Results: We identified segmental paternal UPD in DNA extracted from pancreatic tissue in both patients. UPD was not observed in DNA extracted from the patient's leukocytes or buccal samples. In both cases the UPD encompassed the differentially methylated region at chromosome 11p15.5. Despite this neither patient had any further features of BWS. Conclusion: Paternal UPD of the chromosome 11p15.5 differentially methylated region limited to the pancreatic tissue may represent a novel cause of isolated diazoxide unresponsive HH. Loss of heterozygosity studies should therefore be considered in all patients with severe HH who have undergone pancreatic surgery when K(ATP) channel mutation(s) have not been identified.
 
Biosynthetic pathway for gonadal steroid hormones. (A) Shows known enzymes and intermediates leading to the synthesis of estrogens and androgens from cholesterol. (B) Shows the metabolic steps of dihydrotestosterone processing putatively involved in pre-receptor regulation of ERbeta activation. 3α-Diol = 5α androstane 3α, 17β Diol; 3β Diol = 5α androstane 3β, 17β Diol, RL-HSD = 11-cis-retinol dehydrogenase like 3α-HSD.
E2 and 3β-Diol lead to different patterns of promoter occupancy by ERα and ERβ. mHypoE-38 cells were treated with E2 (10−7 M) or 3β-Diol (10−7 M) for 30 or 60 min. Anti-ERα or anti-ERβ antiserum was used for chromatin immuno-precipitation followed by quantitative PCR of the OT promoter. n = 4; Bars represent the mean ± SEM and are presented as the fold difference of vehicle. **, p < 0.01, ***, p < 0.001.
Activation of the hypothalamo-pituitary-adrenal (HPA) axis is a basic reaction of animals to environmental perturbations that threaten homeostasis. These responses are ultimately regulated by neurons residing within the paraventricular nucleus of the hypothalamus (PVN). Within the PVN, corticotropin-releasing hormone (CRH), vasopressin (AVP) and oxytocin (OT) expressing neurons are critical as they can regulate both neuroendocrine and autonomic responses. Estradiol (E2) and testosterone (T) are well known reproductive hormones, however, they have also been shown to modulate stress reactivity. In rodent models, evidence shows that under some conditions E2 enhances stress activated ACTH and corticosterone secretion. In contrast, T decreases the gain of the HPA axis. The modulatory role of testosterone was originally thought to be via 5 alpha reduction to the potent androgen, dihydrotestosterone, whereas E2 effects were thought to be mediated by both estrogen receptors alpha (ERα) and beta (ERβ). However, DHT has been shown to be metabolized to the ERβ agonist, 5alpha- androstane 3beta,17beta diol (3b-Diol). The actions of 3β-Diol on the HPA axis are mediated by ERbeta which inhibits the PVN response to stressors. In gonadectomized rats, ERbeta agonists reduce CORT and ACTH responses to restraint stress, an effect that is also present in wild-type but not ERbeta knockout mice. The neurobiological mechanisms underlying the actions of ERbeta to alter HPA reactivity are not currently known. CRH, AVP and OT have all been shown to be regulated by estradiol and recent studies indicate an important role of ERbeta in these regulatory processes. Moreover, activation of the CRH and AVP promoters have been shown by 3β-Diol binding to ERbeta and this is thought to be through alternate pathways of gene regulation. Based on available data, a novel and important role for 3beta Diol in the regulation of the HPA axis is suggested.
 
Percentage of 19-norandrosterone glucuronide produced from 1 μM 19-norandrosterone in presence of UGTs specific inhibitors (hecogenin acetate 10 μM for UGT1A4, gemfibrozil 10 μM for UGT2B7, and valproic acid 1 mM for UGT2B15) in human liver microsomes (n = 5) presented as mean ± SD. Significant inhibition was found for the UGT2B7 and UGT2B15 specific inhibitors.
Glucuronidation activity of 19-NA in human liver microsomes in relation to (A) UGT2B7 and (B) UGT2B15 polymorphisms. There was no association between UGT2B7 (H268Y) genotype and glucuronidation activity, whereas a significant association between UGT2B15 (D85Y) polymorphism and glucuronidation activity (ANOVA p = 0.02) was observed.
(A) UGT2B7 and (B) UGT2B15 mRNA expression in HepG2 cells after 2 h incubation with 1 μM 19-NT or testosterone enanthate (n = 8) presented as mean ± SD. UGT2B7 mRNA levels were significantly up-regulated by both androgens, whereas UGT2B15 was significantly induced by testosterone enanthate.
Nandrolone (19-nortestosterone) is an anabolic androgenic steroid commonly abused for doping purposes. Nandrolone is mainly metabolized in the liver into 19-norandrosterone prior to glucuronidation and excretion through urine over an extended period of time. Several UGTs (i.e., UGT2B7, UGT2B15, and UGT2B17) are thought to be the major enzymes responsible for conjugation of androgens in human. An in vitro study using recombinant enzymes expressed in insect cells showed that UGT1A4 and UGT2B7 are the two main enzymes responsible of 19-norandrosterone glucuronidation. However, the identity of the enzyme involved in nandrolone metabolism in vivo together with their relative contribution and regulation remain unknown. Inhibition assays using human liver microsomes (HLM) incubated with 19-norandrosterone and selective inhibitors confirmed that UGT2B7 and UGT2B15 are involved in 19-norandrosterone glucuronidation, since the presence of the specific UGT2B7 and UGT2B15 inhibitors gemfibrozil and valproic acid inhibited the 19-norandrosterone glucuronidation by 35 and 45%, respectively. HLM were genotyped for UGT2B15 D85Y, UGT2B7 H268Y, and the UGT2B17 deletion polymorphism. The glucuronidation activity on 19-norandrosterone was significantly higher in UGT2B15 DD than in the other UGT2B15 genotypes (p < 0.05). Moreover, human liver cancer HepG2 cells were exposed to androgens to determine if the transcriptional activity of the genes of interest was affected. Only UGT2B7 mRNA expression was significantly increased (1.8-folds) after incubation with nandrolone decanoate. These results show that the UGT2B7 and UGT2B15 are involved in 19-norandrosterone glucuronidation and that the UGT2B15 polymorphism (D85Y) is the only UGT genetic variation that influences the glucuronidation activity. This could partly explain the inter-individual variation in 19-norandrosterone excretion.
 
Actions of GLP-1 in peripheral tissues. Most of the effects of GLP-1 are mediated by direct interaction with GLP-1R on specific tissues. However, the actions of GLP-1 in liver, fat, and muscle most likely occur through indirect mechanisms. GLP-1 induces the proliferation of pancreatic duct cells and thyroid C-cells. Reprinted from Gastroenterology (Baggio and Drucker, 2007).
Intracellular signaling pathways of GLP-1R in the pancreatic β-cell. One of the main physiological roles of GLP-1 is to enhance insulin secretion in a glucose-dependent manner. To stimulate insulin secretion and biosynthesis (green), GLP-1R coupled to adenylyl cyclase leading to the activation of cAMP-regulated guanine nucleotide exchange factor II (cAMP-GEFII, also known as Epac2) signaling pathway. GLP-1 plays also a key role in the homeostasis of β-cell mass by inducing β-cell proliferation (blue) and protecting against apoptosis (red). These functions are mediated via the activation of the cAMP/PKA/CREB (cAMP-responsive element binding protein) and the transactivation of the epidermal growth factor receptor (EGF-R) leading to the activation of phosphatidylinositol-3 kinase (PI3K), Protein Kinase Cζ (PKCζ), Akt-protein kinase B, ERK1/2 (Extracellular Regulated Kinase, named also MAPK, Mitogen-Activated Protein Kinase) signaling pathways, and to the up-regulation of the expression of the cell cycle regulator cyclin D1. GLP-1R agonists also improve β-cell function and survival during endoplasmic reticulum stress (purple) by enhancing of ATF-4 translation in a cAMP- and PKA-dependent manner, promoting the up-regulation of the endoplasmic reticulum stress markers CHOP and GADD34 expression and the dephosphorylation of eIF2α. Of note, there is considerable overlap between pathways induced by the GLP-1R activation. Reprinted from Gastroenterology (Baggio and Drucker, 2007).
Cellular signaling mechanisms of GPER and classic nuclear estrogen receptors ERs (↓ activate; ┴ inhibit). ERs are widely accepted as mainly mediating gene transcriptional regulation. Tamoxifen is an ER antagonist in some tissue, such as breast cancer, while has agonistic effects in other tissues, such as endometrium. GPER was found predominantly in the endoplasmic reticulum; estrogen and tamoxifen can bind GPER, and then activate multiple cellular effectors, such as ERK, PI3K, and PLC, and other rapid cellular processes. Most of them are mediated by transactivation of EGF-R. Reprinted from Endocrinology (Wang et al., 2010).
G protein-coupled receptors (GPCRs) constitute a large family of receptors that sense molecules outside the cell and activate inside signal transduction pathways and cellular responses. GPCR are involved in a wide variety of physiological processes, including in the neuroendocrine system. GPCR are also involved in many diseases and are the target of 30% of marketed medicinal drugs. Whereas the majority of the GPCR-targeting drugs have proved their therapeutic benefit, some of them were associated with undesired effects. We develop two examples of used drugs whose therapeutic benefits are tarnished by carcinogenesis risks. The chronic administration of glucagon-like peptide-1 (GLP-1) analogs widely used to treat type-2 diabetes was associated with an increased risk of pancreatic or thyroid cancers. The long-term treatment with the estrogen antagonist tamoxifen, developed to target breast cancer overexpressing estrogen receptors ER, presents agonist activity on the G protein-coupled estrogen receptor which is associated with an increased incidence of endometrial cancer and breast cancer resistance to hormonotherapy. We point out and discuss the need of pharmacological studies to understand and overcome the undesired effects associated with the chronic administration of GPCR ligands. In fact, biological effects triggered by GPCR often result from the activation of multiple intracellular signaling pathways. Deciphering which signaling networks are engaged following GPCR activation appears to be primordial to unveil their contribution in the physiological and physiopathological processes. The development of biased agonists to elucidate the role of the different signaling mechanisms mediated by GPCR activation will allow the generation of new therapeutic agents with improved efficacy and reduced side effects. In this regard, the identification of GLP-1R biased ligands promoting insulin secretion without inducing pro-tumoral effects would offer therapeutic benefit.
 
IL-1β and TNF-α production by mouse calvarial osteoblasts. Osteoblasts in monolayer culture were subjected to a cyclic tensile strain (6 s every 90 s) for 2–48 h and the culture media assayed for IL-1β and TNF-α by ELISAs. Results are expressed as mean ± SEM for 10 cultures. ***Experimental significantly greater than control. P < 0.001.
IL-6 production by mouse calvarial osteoblasts. Osteoblasts in monolayer culture were subjected to a cyclic tensile strain (6 s every 90 s) for 2–48 h and the culture media assayed for IL-6 by an ELISA. Results are mean ± SEM for 10 cultures. ***Experimental significantly greater than control.P < 0.001.
RANKL and OPG production by mouse calvarial osteoblasts. Osteoblasts in monolayer culture were subjected to a cyclic tensile strain (6 s every 90 s) for 2–48 h and the culture media assayed for RANKL and OPG by ELISAs. Results are expressed as mean ± SEM for 10 cultures. ***Experimental significantly different from control.P < 0.001.
Effect of conditioned media from osteoblast cultures on the surface area of mouse osteoclast resorption lacunae. Osteoclasts were obtained from the femurs of 2–3-day-old BALB/C mice and allowed to settle on ivory slices for 20 min at 37°C. The substrate was washed free of non-adherent cells and the slices incubated for 24 h in 500 μL of conditioned medium plus 500 μl fresh DMEM; resorption was quantified by measuring the surface area of the resorption lacunae by image analysis. The values represent the means ± SEM from four slices at each time point. ***Experimental significantly less than control.P < 0.001.
Mechanical strain is an important determinant of bone mass and architecture, and the aim of this investigation was to further understand the role of the cell-cell signaling molecules, IL-1β, TNF-α, and IL-6 in the mechanobiology of bone. Mouse calvarial osteoblasts in monolayer culture were subjected to a cyclic out-of-plane deformation of 0.69% for 6 s, every 90 s for 2-48 h, and the levels of each cytokine plus their downstream targets RANKL and OPG measured in culture supernatants by ELISAs. Mouse osteoblasts constitutively synthesized IL-1β, TNF-α, and IL-6, the production of which was significantly up-regulated in all three by cyclic mechanical strain. RANKL and OPG were also constitutively synthesized; mechanical deformation however, resulted in a down-regulation of RANKL and an up-regulation OPG synthesis. We next tested whether the immunoreactive RANKL and OPG were biologically active in an isolated osteoclast resorption pit assay - this showed that culture supernatants from mechanically deformed cells significantly inhibited osteoclast-mediated resorptive activity across the 48 h time-course. These findings are counterintuitive, because IL-1β, TNF-α, and IL-6 have well-established reputations as bone resorptive agents. Nevertheless, they are pleiotropic molecules with multiple biological activities, underlining the complexity of the biological response of osteoblasts to mechanical deformation, and the need to understand cell-cell signaling in terms of cytokine networks. It is also important to recognize that osteoblasts cultured in vitro are deprived of the mechanical stimuli to which they are exposed in vivo - in other words, the cells are in a physiological default state that in the intact skeleton leads to decreased bone strains below the critical threshold required to maintain normal bone structure.
 
Flowchart of the patients retrospectively included in the study.
Fluctuation of the levels of Testosterone (A) and Estradiol (B) during the first 18 weeks of treatment with Testosterone Undecanoate stratified according to the UGT2B17 genotype. Arrows indicate the three injections.
Background Testosterone (T) is mainly excreted in the urine as testosterone glucuronide (TG). This glucuronidation is partly dependent on the UGT2B17 genotype, and TG excretion is therefore lower in men having the UGT2B17 deletion. However, the possible influence of UGT2B17 genotype on serum T during androgen therapy is unknown. We retrospectively investigated the possible association between the UGT2B17 gene polymorphism and serum T levels in hypogonadal men during Testosterone undecanoate (TU) substitution therapy.Subjects and methods207 patients treated with TU (Nebido®) were genotyped by qPCR for the UGT2B17 deletion polymorphism. All were given 1000 mg TU per injection at 0, 6 and 18 weeks. Blood samples were taken 2 and 6 weeks after the 1st and 2nd injection, prior to the 3rd injection, and after 2-3 years of treatment. We analysed for the levels of T, luteinizing hormone, sex-hormone-binding globulin, estradiol, prostate specific antigen, haematocrit, haemoglobin and total cholesterol. ResultsThe UGT2B17 genotype frequency was: ins/ins: 42%, ins/del: 44% and del/del: 14%. During the initial 18 weeks of TU treatment, large intra- and inter-individual variations in serum T levels were observed. Large peaks in T levels, ranging from 6.7 nmol/l to 69.5 nmol/l, were noted 2 weeks after injections, regardless of the genotype. T levels did not differ between the 3 genotypes prior to the 3rd injection, but the del/del group had significantly lower levels of LH. At follow-up after 2-3 years, the injection interval or daily T dosage was not dependent on the UGT2B17 genotype. Conclusion In conclusion, we found large intra- and inter-individual variations in serum T during standard TU treatment regimen in hypogonadal men. Only subtle differences in serum T and LH were noted according to UGT2B17 genotype, which however suggest that the UGT2B17 genotype exert modest influence on the pharmacokinetic profile of T after TU treatment.
 
Digit ratio (2D:4D) denotes the relative length of the second and fourth digits. This ratio is considered to be a biomarker of the balance between fetal testosterone (T) and estrogen (E) in a narrow window of early ontogeny. Evidence for this assertion is derived from direct and indirect measures of prenatal hormonal exposure (in experimental animals, via amniotic fluid samples and in the study of sex-typical traits) in relation to 2D:4D. In contrast, the relationships between 2D:4D and levels of sex steroids in adults are less clear, as many correlational studies of 2D:4D and adult sex steroids have concluded that this association is statistically non-significant. Here, we suggest that in order to understand the link between 2D:4D and sex hormones, one must consider both fetal organizing and adult activating effects of T and E. In particular, we hypothesize that 2D:4D correlates with organizing effects on the endocrine system that moderate activating effects in adulthood. We argue that this is particularly evident in "challenging" conditions such as aggressive and sexual encounters, in which individuals show increased levels of T. We discuss this refinement of the 2D:4D paradigm in relation to the links between 2D:4D and sports performance, and aggression.
 
Fictitious example of in vitro study into the effect of CAGn on target gene activity. Example for regression of the activity of a testosterone regulated target gene on CAGn in in vitro studies (cf. Table 1). ARs with CAGn of 0, 15, 18, 21, 24, or 27 are used either with testosterone (gray bars) or without testosterone (black bars). Target gene activity observed at CAGn most typical in humans (21) is set to 100%. Then the regression slope (dashed line, −2.3%) is calculated to describe target gene activity as a function of CAGn. In cases like the present, where CAGn outside the human range produce a deviation from linearity (here 0 CAGn), the regression slope was calculated only for those CAGn that showed a linear function.
| Change in androgen driven target gene activity per additional CAG repeat in the AR gene in in vitro studies.
| Primary studies investigating the relationship between 2D:4D and CAGn.
| Results of meta-analyses for the relationship between CAGn and 2D:4D.
The length ratio of the second to the fourth digit (2D:4D) is a putative marker of prenatal testosterone (T) effects. The number of CAG repeats (CAGn) in the AR gene is negatively correlated with T sensitivity in vitro. Results regarding the relationship between 2D:4D and CAGn are mixed but have featured prominently in arguments for and against the validity of 2D:4D. Here, I present random-effects meta-analyses on 14 relevant samples with altogether 1904 subjects. Results were homogeneous across studies. Even liberal estimates (upper limit of the 95% CI) were close to zero and therefore suggested no substantial relationship of CAGn with either right-hand 2D:4D, left-hand 2D:4D, or the difference between the two. However, closer analysis of the effects of CAGn on T dependent gene activation in vitro and of relationships between CAGn and T dependent phenotypic characteristics suggest that normal variability of CAGn has mostly no, very small, or inconsistent effects. Therefore, the lack of a clear association between CAGn and 2D:4D has no negative implications for the latter's validity as a marker of prenatal T effects.
 
| Biochemical and anthropometric characteristics of children with signs of premature adrenarche and those of prepubertal control children.
| Genotype frequencies of the TNF-alpha -308 polymorphism in children with signs of premature adrenarche (PA) and in control children.
Premature adrenarche (PA), the early rise in adrenal androgen production leading to prepubertal signs of androgen action, has been connected with adverse metabolic features. The metabolic syndrome is characterized by low-grade inflammation which in turn is associated with increases in circulating proinflammatory cytokines, like tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). We tested the hypothesis that serum concentrations of TNF-α and IL-6 are increased in PA by studying 73 children with PA and 98 age- and gender-matched controls. Serum TNF-α and IL-6 concentrations were measured using a multiplex bead array. The subjects were genotyped for the TNF-α gene -308 G > A polymorphism (known to affect TNF-α gene transcription), and genotype-phenotype associations were studied. The mean serum TNF-α concentration was higher in the PA than control children (20.4 vs. 18.4 pg/ml, P = 0.048), whereas there was no significant difference in the mean serum IL-6 concentrations between the study groups. The difference in TNF-α was not explained by excess body weight in the PA subjects as the difference remained significant after BMI-adjustment (P = 0.038). In the PA group, TNF-α concentration was not associated with metabolic-endocrine features, but high IL-6 was associated with lower birth weight. There was no difference in the genotype distribution of the TNF-α gene -308 G > A polymorphism between the PA and control groups. In conclusion, PA was associated with increased serum TNF-α concentrations which, unexpectedly, were not connected with BMI or insulin resistance. The TNF-α gene -308 G > A polymorphism does not seem to be associated with the development of PA.
 
Ellipsoids of differing proportions. (A) In curved geometries (gray), prolate [javelin-shaped, yellow, (B)] ellipsoids maximally fit rod-like regions, intermediate ellipsoids [red, (C)] maximally fit junction regions, and oblate [discus-shaped, purple, (D)] ellipsoids maximally fit plate-like regions. Ellipsoids have three semi-axes (radii), a, b, and c (C). Prolate ellipsoids (B) have one long radius and two short radii such that a ≤ b ≪ c, while oblate ellipsoids (D) have two long radii and one short radius such that a ≪ b ≤ c. Intermediate ellipsoids (C) have more moderately differing radii, a ≤ b ≤ c. The ellipsoid factor (EF) of an ellipsoid is calculated as EF = a/b − b/c.
| Comparison of ellipsoid factor to SMI, BV/TV, and Tb.Th.
Results of BoneJ’s ellipsoid factor implementation, run on X-ray microtomographic images of trabecular bone from the femora of emu [Dromaius novaehollandiae, (A–D)] and lesser dwarf shrew [Suncus varilla (E–H)], and on a synthetic image of rods and plates (I–L). Input geometry (A,E,I) was processed using default settings, except that all skeleton points were used (default is to use only every 50th point). 3D color map images (B,F,J) indicate EF > 0 in orange–yellow and EF < 0 in purple–blue [look-up table is the same as in (D,H,L)]. Note the labeling of rods in orange–yellow and plates in purple–blue. Flinn diagrams (C,G,K) demonstrate the distribution of axis ratios toward the top left for rod-dominated structures (G) and the bottom right for plate-dominated structures (C). (K) shows discrete clusters of peaks relating to the rods and plates in the synthetic image. The diagonal indicates the line where a/b = b/c; ellipsoids are spherical at the top right corner. Histograms and summary statistics of EF (D,H,L) display a shift to the left for plate-dominated structures (D), to the right for rod-dominated structures (H) and a bimodal distribution for structures with few intermediate ellipsoids (L).
The ellipsoid factor (EF) is a method for the local determination of the rod- or plate-like nature of porous or spongy continua. EF at a point within a 3D structure is defined as the difference in axis ratios of the greatest ellipsoid that fits inside the structure and that contains the point of interest, and ranges from -1 for strongly oblate (discus-shaped) ellipsoids, to +1 for strongly prolate (javelin-shaped) ellipsoids. For an ellipsoid with axes a ≤ b ≤ c, EF = a/b - b/c. Here, EF is demonstrated in a Java plugin, "Ellipsoid Factor" for ImageJ, distributed in the BoneJ plugin collection. Ellipsoid Factor utilizes an ellipsoid optimization algorithm, which assumes that maximal ellipsoids are centered on the medial axis, then dilates, rotates, and translates slightly each ellipsoid until it cannot increase in volume any further. EF successfully identifies rods, plates, and intermediate structures within trabecular bone, and summarizes the distribution of geometries with an overall EF mean and SD, EF histogram, and Flinn diagram displaying a/b versus b/c. EF is released to the community for testing, use, and improvement.
 
Melanin-concentrating hormone receptor 1 (MCHR1) is a G-protein-coupled receptor (GPCR) that plays an important role in feeding by coupling to Gα(q)- and Gα(i)-mediated signal transduction pathways. To interrogate the molecular basis for MCHR1 activation, we analyzed the effect of a series of site-directed mutations on rat MCHR1 function. In the highly conserved NPxxY(x)(5,6)F domain of GPCRs, the phenylalanine residue is involved in structural constraints; replacement with alanine generally leads to impaired/lost GPCR function. However, Phe-to-Ala (F318A) mutation in MCHR1 had no significant effect on the level of cell surface expression and receptor signaling. By analyzing a further series of mutants, we found that Phe-to-Lys substitution (F318K) caused the most significant reduction in the EC(50) value of MCH for calcium mobilization without affecting receptor expression at the cell surface. Interestingly, GTPγS-binding, which monitors Gα(i) activation, was not modulated by F318K. Our results, combined with computer modeling, provide new insight into the role of Phe in the NPxxY(x)(5,6)F motif as a structurally critical site for receptor dynamics and a determinant of Gα protein interaction.
 
Fractures and season of birth.
Vitamin D status in pregnant women has been linked to childhood bone mineral density in their offspring but it is unclear if effects extend to fracture risk in adulthood or even old age. As vitamin D levels in the population show pronounced seasonal variation in Denmark, we performed an epidemiological analysis of hip fracture rates as a function of season of birth, age, and sex. We retrieved information on all hip fractures in the 9-year period between 1997 and 2005 in all men and women aged 65-95, excluded hip fractures that occurred in current and recent prednisolone users, and subsequently calculated fracture rates and relative risks. The analysis covered 541,109 men and 691,522 women. In women, we observed a small but statistically significant difference between fracture rates by season of birth for all age intervals expect the youngest (age 65-69). A similar pattern was seen in men, but this was only statistically significant in the two oldest age groups (age 85-89 and 90-95). These findings suggest that vitamin D availability in the first and second trimester of intrauterine life could have a small but lasting impact on bone health and the risk of osteoporotic fractures. Further studies are needed.
 
| Characteristics of the 15 reclassified cases.
Location of carcinoids within the lungs.
ROC analysis for Ki-67.
Disease-free survival (DFS) in patients with Ki-67 expression more and less than 4%.
Disease-free survival (DFS) in patients with typical (TC) and atypical carcinoid (AC).
Introduction: Histological distinction between typical and atypical bronchopulmonary carcinoids is based on mitotic activity and necrosis. Regardless of these two parameters, outcome after surgery is often unpredictable. In this study the prognostic value of different clinico-pathological factors was retrospectively analyzed in a large series of patients with bronchopulmonary carcinoid. Materials and methods: The long-term post-surgical outcome of 106 radically treated patients affected by bronchopulmonary carcinoid from two Italian centers was correlated with tumor characteristics assessed by combining conventional histology with a panel of immunohistochemical markers of neuroendocrine differentiation (chromogranin-A, NSE) and proliferation activity (Ki-67 score). Results: Carcinoids were assessed as typical (TC = 75; 70.8%) and atypical (AC = 31; 29.2%). Mean follow-up was 8.3 years (range: 0-20; median: 8.0). All cases expressed neuroendocrine markers. At univariate analysis, tumor recurrence [14/75 TC (18.7%), 15/31 AC (48.4%)] correlated with carcinoid histotype (P = 0.003), tumor size (P = 0.012), mitotic index (P = 0.044), Ki-67 score (P < 0.0001), and synchronous node metastasis (P = 0.037). Of these, Cox multivariate analysis confirmed only Ki-67 score as independent predictor of disease recurrence (P = 0.009). The best cut-off for Ki-67 score (calculated by ROC curves) discriminating recurrent vs non-recurrent disease was 4% (sensitivity 79.3%; specificity 83.8%; area under the curve 0.85). By stratifying patients according to this cut-off, a significantly different disease-free survival was found (log-rank test P < 0.0001). Conclusion: Ki-67 score accurately separates bronchopulmonary carcinoids in two well-distinct histo-prognostic categories. Ki-67 score predicts the patients outcome better than mitotic count, histotype, and tumor stage and it is therefore helpful in establishing the appropriate follow-up.
 
ACTH [(A); picogram/milliliter], corticosterone [(B); CORT in nanogram/milliliter] blood plasma levels, and their ratio [(C); ACTH/CORT] of non-deprived (NON-DEP) and 24 h deprived (DEP) pups measured at basal conditions (basal; white bars) or after 30 min of novelty exposure (novelty; black bars) at postnatal day (pnd) 5. Data represent mean ± SEM. * vs. basal, # vs. NON-DEP, $ vs. CD1. Significance level was set at p = 0.05.
CRH (A) and GR (B) mRNA expression in the paraventricular nucleus of the hypothalamus measured in non-deprived (NON-DEP) and deprived (DEP) mice at postnatal day (pnd) 5. Data represent mean ± SEM. # vs. NON-DEP, $ vs. CD1. Significance level was set atp = 0.05.
The postnatal development of the mouse is characterized by a period of hypo-responsiveness of the hypothalamic-pituitary-adrenal (HPA) axis to mild stressors. Maternal deprivation (MD) during this period can disrupt the quiescence of the HPA-axis. The present study examined the influence of strain (outbred CD1 vs. inbred C57BL/6J mice) on some central and peripheral components of the HPA-axis in neonatal mice (5-day-old) in the presence of their mother or after 24 h MD (on postnatal day 4) under basal or mild stressful conditions. In the presence of the dam, adrenal corticosterone (CORT) secretion was low in both mouse strains. Compared to CD1 mice, C57BL/6J had lower CORT levels associated with higher ACTH levels and ACTH/CORT ratio (i.e., lower adrenal sensitivity to ACTH), and higher glucocorticoid receptor (GR) mRNA expression in the paraventricular nucleus. Although MD disinhibited the HPA-axis in both strains as reflected by increased basal CORT and ACTH, we found a strain-dependent pattern. MD increased CORT more in C57BL/6J compared to CD1 mice together with a lower ACTH/CORT ratio (i.e., higher adrenal sensitivity to ACTH), while GR mRNA was no longer different in the two strains. However, this increased adrenal sensitivity in maternally deprived C57BL/6J mice was not reflected in their CORT response to a subsequent novelty stressor, possibly due to an MD-induced ceiling effect in their steroidogenic capacity. In conclusion, the immediate outcome of MD depends on the genetic background of the mother-infant dyad, suggesting that maybe also the outcome in later-life cannot be generalized.
 
Ethanol (EtOH)–induced impairment of long-term potentiation (LTP) in the rat hippocampus is prevented by the 5α-reductase inhibitor finasteride, suggesting that this effect of EtOH is dependent on the increased local release of neurosteroids such as 3α,5α-THP that promote GABA–mediated transmission. Given that social isolation (SI) in rodents is associated with altered plasma and brain levels of such neurosteroids as well as with an enhanced neurosteroidogenic action of EtOH, we examined whether the inhibitory effect of EtOH on LTP at CA3-CA1 hippocampal excitatory synapses is altered in C57BL/6J mice subjected to SI for 6 weeks in comparison with group-housed (GH) animals. Extracellular recording of fEPSPs as well as patch-clamp analysis were performed in hippocampal slices prepared from both SI and GH mice. Consistent with previous observations, recording of fEPSPs revealed that the extent of LTP induced in the CA1 region of SI mice was significantly reduced compared with that in GH animals. EtOH (40 mM) inhibited LTP in slices from SI mice but not in those from GH mice, and this effect of EtOH was abolished by co-application of 1 µM finasteride. Current-clamp analysis of CA1 pyramidal neurons revealed a decrease in action potential frequency and an increase in the intensity of injected current required to evoke the first action potential in SI mice compared with GH mice, indicative of a decrease in neuronal excitability associated with SI. Together, our data suggest that SI results in reduced levels of neuronal excitability and synaptic plasticity in the hippocampus. Furthermore, the increased sensitivity to the neurosteroidogenic effect of EtOH associated with SI likely accounts for the greater inhibitory effect of EtOH on LTP in SI mice. The increase in EtOH sensitivity induced by SI may be important for the changes in the effects of EtOH on anxiety and on learning and memory associated with the prolonged stress attributable to social isolation.
 
Steroids synthesized de novo by the central and peripheral nervous systems are called neurosteroids. The formation of neurosteroids from cholesterol in the brain was originally demonstrated in mammals by Baulieu and colleagues. Our studies over the past two decades have also shown that, in birds and amphibians as in mammals, the brain expresses several kinds of steroidogenic enzymes and produces a variety of neurosteroids. Thus, de novo neurosteroidogenesis from cholesterol is a conserved property that occurs throughout vertebrates. However, the biosynthetic pathways of neurosteroids in the brain of vertebrates was considered to be still incompletely elucidated. Recently, 7α-hydroxypregnenolone was identified as a novel bioactive neurosteroid stimulating locomotor activity in the brain of newts and quail through activation of the dopaminergic system. Subsequently, diurnal and seasonal changes in synthesis of 7α-hydroxypregnenolone in the brain were demonstrated. Interestingly, melatonin derived from the pineal gland and eyes regulates 7α-hydroxypregnenolone synthesis in the brain, thus inducing diurnal locomotor changes. Prolactin, an adenohypophyseal hormone, regulates 7α-hydroxypregnenolone synthesis in the brain, and may also induce seasonal locomotor changes. This review highlights the identification, mode of action, and functional significance of 7α-hydroxypregnenolone, a new key regulator of locomotor activity of vertebrates, in terms of diurnal and seasonal changes in 7α-hydroxypregnenolone synthesis, and describes some of their regulatory mechanisms.
 
ATP-sensitive K+ (KATP) channels composed of potassium inward-rectifier type 6.2 and sulfonylurea receptor type 1 subunits (Kir6.2/SUR1)4 are expressed in various cells in the brain and endocrine pancreas where they couple metabolic status to membrane potential. In β-cells, increases in cytosolic [ATP/ADP]c inhibit KATP channel activity, leading to membrane depolarization and exocytosis of insulin granules. Mutations in ABCC8 (SUR1) or KCNJ11 (Kir6.2) can result in gain or loss of channel activity and cause neonatal diabetes (ND) or congenital hyperinsulinism (CHI), respectively. SUR1 is reported to be a Mg2+-dependent ATPase. A prevailing model posits that ATP hydrolysis at SUR1 is required to stimulate openings of the pore. However, recent work shows nucleotide binding, without hydrolysis, is sufficient to switch SUR1 to stimulatory conformations. The actions of nucleotides, ATP and ADP, on ND (SUR1E1506D) and CHI (SUR1E1506K) mutants, without Kir6.2, were compared to assess both models. Both substitutions significantly impair hydrolysis in SUR1 homologues. SUR1E1506D has greater affinity for MgATP than wildtype; SUR1E1506K has reduced affinity. Without Mg2+, SUR1E1506K has a greater affinity for ATP4- consistent with electrostatic attraction between ATP4-, unshielded by Mg2+, and the basic lysine. Further analysis of ND and CHI ABCC8 mutants in the second transmembrane and nucleotide binding domains (TMD2 & NBD2), found a relation between their affinities for ATP (± Mg2+) and their clinical phenotype. Increased affinity for ATP is associated with ND; decreased affinity with CHI. In contrast, MgADP showed a weaker relationship. Diazoxide, known to reduce insulin release in some CHI cases, potentiates switching of CHI mutants from non-stimulatory to stimulatory states consistent with diazoxide stabilizing a nucleotide-bound conformation. The results emphasize the greater importance of nucleotide binding vs hydrolysis in the regulation of KATP channels in vivo.
 
| Comparison of mean ages of methylation-positive patients for the indicated genes between the ever-smoker and never-smoker groups.
| Comparison of methylation levels for the indicated genes in papillary thyroid cancer between ever and never smokers.
Aberrant gene methylation in papillary thyroid cancer in ever and never smokers. Shown on y-axis is the methylation rate – percentage of the methylation-positive cases (%) – for each of the four genes indicated on the x-axis. *The methylation rate of the RARβ2 gene is significantly higher (P = 0.003) in the ever smoker group than the never smoker group. The methylation rate for the remaining three genes shows a higher trend for ever smokers but does not reach statistical significance.
Aberrant gene methylation is often seen in thyroid cancer, a common endocrine malignancy. Tobacco smoking has been shown to be associated with aberrant gene methylation in several cancers, but its relationship with gene methylation in thyroid cancer has not been examined. In the present study, we investigated the relationship between smoking of patients and aberrant methylation of tumor suppressor genes for TIMP3, SLC5A8, death-associated protein kinase, and retinoic acid receptor β2 (RARβ2) in papillary thyroid cancer (PTC), the most common type of thyroid cancer. The promoter methylation status of these genes was analyzed using quantitative real-time methylation-specific PCR on bisulfite-treated genomic DNA isolated from tumor tissues and correlated with smoking history of the patients. Among the four genes, methylation of the RARβ2 gene was significantly associated with smoking and other three genes showed a trend of association. Specifically, among the 138 patients investigated, 13/42 (31.0%) ever smokers vs. 10/96 (10.4%) never smokers harbored methylation of the RARβ2 gene (P = 0.003). This association was highly significant also in the subset of conventional variant PTC (P = 0.005) and marginally significant in follicular variant PTC (P = 0.06). The results demonstrate that smoking-associated aberrant methylation of the RARβ2 gene is a specific molecular event that may represent an important mechanism in thyroid tumorigenesis in smokers.
 
Metabolic shifts through the hexosamine biosynthesis pathway (HBP) and protein O-GlcNAcylation in cancer. The HBP produces UDP-GlcNAc from its parts, glucose (Glc), glutamine (Gln), acetyl-CoA, and UTP. The levels of these various metabolic inputs are all increased in cancer cells. Glucose is transported into cells by glucose transporters (e.g., GLUT1). Overexpression or mutation of c-Myc and Kras leads to an increase of glucose uptake through GLUT1 activation. O-GlcNAcylation of PFK1 suppresses the enzyme activity, resulting in redirection of glucose metabolism. GFAT is the rate-limiting enzyme for glucose entry into the HBP, which converts Fruc-6-P and glutamine (Gln) into GlcN-6-P. Hypoxia induces the GFAT transcription and expression. Glucosamine (GlcN) enters into cells via the glucose transporters and is phosphorylated to GlcN-6-P by hexokinase, bypassing GFAT. UDP-GlcNAc serves as a sugar donor of classical glycosylation and O-GlcNAcylation. The later glycosylation, taking place in cytoplasm, nucleus, and mitochondria, is controlled by O-GlcNAc cycling enzymes; OGT and OGA for the addition and removal of sugar in and out of proteins, respectively. OGT level is also upregulated, and consequently results in an increase of O-GlcNAcylation in several kinds of cancers.
O-GlcNAcylated proteins and their targets identified in colorectal cancer. O-GlcNAcylation stabilizes β-catenin and subsequently translocates into the nucleus for its gene activation. O-GlcNAcylation stabilizes Snail1, which subsequently represses E-cadherin expression level observed in other cancer cells, suggesting a proposed mechanism for colorectal cancer. Phosphorylation of β-catenin and Snail1 is proposed to activate proteasomal degradation. The proposed mechanisms for other O-GlcNAcylated proteins, including SP1, CK18, α-tubulin, hnRNPA2/B1, hnRNPH, annexin A2, annexin A7, laminin-binding protein, and protein DJ-1, are also indicated. Expression levels of E-cadherin, β-catenin, caveolin-1, and IκB-β are altered corresponding to increased global O-GlcNAcylation, so they are categorized as O-GlcNAc downstream targets. Solid lines indicate known mechanisms, whereas dashed lines are proposed mechanisms.
O-GlcNAcylated proteins and their targets identified in breast cancer. O-GlcNAcylation modifies many proteins in both the cytoplasm and nucleus. p120, β-catenin, and E-cadherin are glycosylated, and this modification regulates E-cadherin localization and stability. O-GlcNAc Snail1 suppresses E-cadherin expression. Phosphorylation of β-catenin and Snail1 can activate proteasomal degradation while O-GlcNAc p53 protects this event. Other cytoskeletal proteins (cofilin, β-actin, vimentin, keratin 7, annexin A2, and CK18), glycolytic enzymes (PKF1, PKM2, GAPDH, TPI, ENO2, and ENO1), chaperones (protein DJ1, HSC70, HSP27, PDIA6, peroxiredoxin 3, 4, and 6), thymidine phosphorylase, and VDAC1 are also O-GlcNAcylated. Nuclear proteins including hnRNPs and proteins related to transcription and translation are O-GlcNAcylated as indicated. Alteration of this modification also regulates gene and protein expressions in breast cancer including FoxM1 and EZH2. Solid lines indicate known mechanisms, whereas dashed lines are proposed mechanisms.
Increasing glucose consumption is thought to provide an evolutionary advantage to cancer cells. Alteration of glucose metabolism in cancer influences various important metabolic pathways including the hexosamine biosynthesis pathway (HBP), a relatively minor branch of glycolysis. Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), an end product of HBP, is a sugar substrate used for classical glycosylation and O-GlcNAcylation, a post-translational protein modification implicated in a wide range of effects on cellular functions. Emerging evidence reveals that certain cellular proteins are abnormally O-GlcNAc modified in many kinds of cancers, indicating O-GlcNAcylation is associated with malignancy. Since O-GlcNAc rapidly on and off modifies in a similar time scale as in phosphorylation and these modifications may occur on proteins at either on the same or adjacent sites, it suggests that both modifications can work to regulate the cellular signaling pathways. This review describes the metabolic shifts related to the HBP, which are commonly found in most cancers. It also describes O-GlcNAc modified proteins identified in primary breast and colorectal cancer, as well as in the related cancer cell lines. Moreover, we also discuss the potential use of aberrant O-GlcNAcylated proteins as novel biomarkers of cancer.
 
| Brain MR imaging in a 1-year-old girl with transient CHI, showing occipital lobe atrophy (OLA) and periventricular high signal intensities (PVHSI) in sagittal T2 FLAIR sequence scanning (1a) and
Brain MR imaging in a 1-year-old girl with transient CHI, showing occipital lobe atrophy (OLA) and periventricular high signal intensities (PVHSI) in sagittal T2 FLAIR sequence scanning (1a) and occipital hyperintense signals (OHS) within subcortical white matter in proton density and T2 axial images, the latter being highly suggestive of hypoglycemic injury.
The severity of abnormal neurodevelopment in patients with CHI. Mild and severe abnormal neurodevelopment for both persistent (P-CHI) and transient CHI (T-CHI); the prevalence of severe abnormal neurodevelopment is similar in both groups, implying that early severity, but not the duration of hypoglycemia is important in determining the outcome of hypoglycemia in children with CHI.
Introduction: Neuroglycopenia is recognized to be associated with abnormal neurodevelopmental outcomes in 26–44% of children with persistent congenital hyperinsulinism (P-CHI). The prevalence of abnormal neurodevelopment in transient CHI (T-CHI) is not known. We have aimed to investigate abnormal neurodevelopment and associated factors in T-CHI and P-CHI. Materials and Methods: A cohort of children with CHI (n = 67, age 2.5–5 years) was assessed at follow-up review and noted to have normal or abnormal (mild or severe) neurodevelopmental outcomes for the domains of speech and language, motor, and vision. Children were classified as P-CHI (n = 33), if they had undergone surgery or remained on medical therapy, or T-CHI (n = 34), if medical treatment for hypoglycemia was stopped. Results: Overall, abnormal neurodevelopment was present in 26 (39%) children with CHI, of whom 18 (69%) were severe. Importantly, the incidence of abnormal neurodevelopment in T-CHI was similar to that in P-CHI (30 vs. 47% respectively, p = 0.16). The prevalence of severe abnormal neurodevelopment in speech, motor, and vision domains was similar in both T-CHI and P-CHI children. For this cohort, we found that the severity of disease [based upon maximal diazoxide dose (odds ratio 95% confidence intervals) 1.3 (1.1; 1.5), p = 0.03], and early presentation of CHI <7 days following birth [5.9 (1.3; 27.8), p = 0.02] were significantly associated with abnormal neurodevelopment. There was no significant association with gender, genotype, or the histopathological basis of CHI. Conclusion: Abnormal neurodevelopment was evident in one third of children with both T-CHI and P-CHI, early presentation and severe CHI being risk factors. Early recognition and rapid correction of hypoglycemia are advocated to avoid abnormal neurodevelopment in children with CHI.
 
Cascade of effects originating in neurodevelopmental insults occurring at different points in development. Insults can occur at any point in neurodevelopment from embryonic through to juvenile and perhaps even adulthood. Insults to neural crest and neural tube development in the embryo would have structural and functional outcomes in all peripheral neural elements including autonomic (sympathetic, parasympathetic), peripheral sensory system, and enteric (gut) nervous system. Neural tube effects might also produce changes in central autonomic functions and/or set points for physiological control systems resident in the hypothalamus and brain stem autonomic nuclei. Factors affecting cortical development would have an impact on higher cortical functions including psychosocial, sensory processing, and motor functioning. Any of these neurodevelopmental effects would have cascading effects through the sensory and neuro-immune pathways to neurological, neuro-immune, and immune functions.
| Causes of death in ASD with moderate to severe retardation or none to mild retardation (in brackets).
| Broad clinical sub-categories of ASD.
Autism spectrum disorder (ASD) is a heterogeneous condition affecting an individual's ability to communicate and socialize and often presents with repetitive movements or behaviors. It tends to be severe with less than 10% achieving independent living with a marked variation in the progression of the condition. To date, the literature supports a multifactorial model with the largest, most detailed twin study demonstrating strong environmental contribution to the development of the condition. Here, we present a brief review of the neurological, immunological, and autonomic abnormalities in ASD focusing on the causative roles of environmental agents and abnormal gut microbiota. We present a working hypothesis attempting to bring together the influence of environment on the abnormal neurological, immunological, and neuroimmunological functions and we explain in brief how such pathophysiology can lead to, and/or exacerbate ASD symptomatology. At present, there is a lack of consistent findings relating to the neurobiology of autism. Whilst we postulate such variable findings may reflect the marked heterogeneity in clinical presentation and as such the variable findings may be of pathophysiological relevance, more research into the neurobiology of autism is necessary before establishing a working hypothesis. Both the literature review and hypothesis presented here explore possible neurobiological explanations with an emphasis of environmental etiologies and are presented with this bias.
 
Dietary treatment with high-fat diets (HFD) triggers diabetes and hyperleptinemia, concomitantly with a partial state of leptin resistance that affects hepatic and adipose tissue but not the heart. In this context, characterized by widespread steatosis, cardiac lipid content remains unchanged. As previously reported, HFD-evoked hyperleptinemia could be a pivotal element contributing to increase fatty-acid (FA) metabolism in the heart and to prevent cardiac steatosis. This metabolic adaptation might theoretically reduce energy efficiency in cardiomyocytes and lead to cardiac electrophysiological remodeling. Therefore the aim of the current study has been to investigate the impact of long-term HFD on cardiac metabolism and electrophysiological properties of the principal ionic currents responsible of the action potential duration in mouse cardiomyocytes. Male C57BL/6J mice were fed a control (10 kcal% from fat) or HFD (45 kcal% from fat) during 32 weeks. Quantification of enzymatic activities regulating mitochondrial uptake of pyruvate and FA showed an increase of both carnitine-palmitoyltransferase and citrate synthase activities together with a decrease of lactate dehydrogenase and pyruvate dehydrogenase activities. Increased expression of uncoupling protein-3, Mn-, and Cu/Zn-superoxide dismutases and catalase were also detected. Total glutathione/oxidized glutathione ratios were unaffected by HFD. These data suggest that HFD triggers adaptive mechanisms aimed at (i) facilitating FA catabolism, and (ii) preventing oxidative stress. All these changes did not affect the duration of action potentials in cardiomyocytes and only slightly modified electrocardiographic parameters.
 
Confocal images (1-μm optical section) of a tissue section through the arcuate nucleus processed for triple-label immunofluorescent detection of kisspeptin (Kiss), neuropeptide Y (NPY), and synaptophysin (Syn). Immunoreactive kisspeptin soma and dendrites (blue) are observed in close proximity to NPY- [gren; (A)] and synaptophysin- [red; (B)] positive axons. (C) Synaptophysin colocalization in NPY axons that are in close proximity to kisspeptin neurons. Arrows indicate examples of NPY/synaptophysin-positive terminals. Scale bar, 20 μm.
Model for pathways mediating the nutritional regulation of GnRH release during pubertal development. Signals of nutrient sufficiency such as hormones (e.g., leptin) and metabolites are perceived by metabolic-sensing neurons in the hypothalamus (e.g., NPY/AgRP and POMC neurons) that project directly to GnRH neuron soma and dendrites, and/or terminals in the median eminence (ME; not represented). NPY/AgRP and POMC neurons may also regulate GnRH neurons indirectly via kisspeptin neurons. Neurons in the premammillary nucleus could also represent a leptin-sensitive pathway for regulation of GnRH neurons during pubertal transition, but neuronal phenotype and hypothetical projections (green dashed lines) are yet to be characterized. Direct action of leptin on kisspeptin neurons (black dashed line) is unlikely to represent a major pathway. Accelerated growth and adiposity during the juvenile period hastens the peripubertal activation of GnRH neurons by reducing inhibitory signals (e.g., NPY) and enhancing stimulatory signals (e.g., kisspeptin, POMC-derived peptides), and leads to increased frequency of episodic release of GnRH and early onset of puberty.
The pubertal process is characterized by an activation of physiological events within the hypothalamic-adenohypophyseal-gonadal axis which culminate in reproductive competence. Excessive weight gain and adiposity during the juvenile period is associated with accelerated onset of puberty in females. The mechanisms and pathways by which excess energy balance advances puberty are unclear, but appear to involve an early escape from estradiol negative feedback and early initiation of high-frequency episodic gonadotropin-releasing hormone (GnRH) secretion. Hypothalamic neurons, particularly neuropeptide Y and proopiomelanocortin neurons are likely important components of the pathway sensing and transmitting metabolic information to the control of GnRH secretion. Kisspeptin neurons may also have a role as effector neurons integrating metabolic and gonadal steroid feedback effects on GnRH secretion at the time of puberty. Recent studies indicate that leptin-responsive neurons within the ventral premammillary nucleus play a critical role in pubertal progression and challenge the relevance of kisspeptin neurons in this process. Nevertheless, the nutritional control of puberty is likely to involve an integration of major sensor and effector pathways that interact with modulatory circuitries for a fine control of GnRH neuron function. In this review, observations made in ruminant species are emphasized for a comparative perspective.
 
Immigrants to a new country face many challenges when diagnosed with type 2 diabetes, a chronic disease with a complex treatment involving both medical and behavioral interventions. These challenges will depend upon the extent to which the patient has adapted to the new country's social and cultural norms, as well as individual factors such as age, education, and gender. This adaptation is termed acculturation. With respect to nutritional interventions for type 2 diabetes, uptake and adherence over the long term will depend upon overall health literacy, the cultural acceptability of the recommended diet. This review has focused on acculturation and its effects on diabetes management in ethnic Chinese in North America as an example of one populous minority and the challenges faced in adopting nutritional recommendations. Research directions and practical considerations are suggested.
 
Schematic representation of the domain structure of MRAP highlighting regions of functional significance in MRAP dimerization and in its interaction with MC2R (Sebag and Hinkle, 2009b; Webb et al., 2009).
Schematic representation of the role of MRAP in MC2R signaling. MRAP monomers form unique anti-parallel homodimeric complexes in the endoplasmic reticulum which interact with MC2R and assist its trafficking to the cell surface. The MRAP/MC2R structure is capable of binding ACTH at the cell surface and initiating intracellular signaling.
The five melanocortin receptors (MCRs) named MC1R-MC5R have diverse physiological roles encompassing pigmentation, steroidogenesis, energy homeostasis and feeding behavior as well as exocrine function. Since their identification almost 20 years ago much has been learnt about these receptors. As well as interacting with their endogenous ligands the melanocortin peptides, there is now a growing list of important peptides that can modulate the way these receptors signal, acting as agonists, antagonists, and inverse agonists. The discovery of melanocortin 2 receptor accessory proteins as a novel accessory factor to the MCRs provides further insight into the regulation of these important G protein-coupled receptor.
 
Inflammatory adipokines secreted from adipose tissue are major contributors to obesity-associated inflammation and other metabolic dysfunctions. We and others have recently documented the contribution of adipose tissue renin-angiotensin system to the pathogenesis of obesity, inflammation, and insulin resistance. We hypothesized that adipocyte-derived angiotensinogen (Agt) plays a critical role in adipogenesis and/or lipogenesis as well as inflammation. This was tested using 3T3-L1 adipocytes, stably transfected with Agt-shRNA or scrambled Sc-shRNA as a control. Transfected preadipocytes were differentiated and used to investigate the role of adipose Agt through microarray and PCR analyses and adipokine profiling. As expected, Agt gene silencing significantly reduced the expression of Agt and its hormone product angiotensin II (Ang II), as well as lipid accumulation in 3T3-L1 adipocytes. Microarray studies identified several genes involved in lipid metabolism and inflammatory pathways which were down-regulated by Agt gene inactivation, such as glycerol-3-phosphate dehydrogenase 1 (Gpd1), serum amyloid A 3 (Saa3), nucleotide-binding oligomerization domain containing 1 (Nod1), and signal transducer and activator of transcription 1 (Stat1). Mouse adipogenesis PCR arrays revealed lower expression levels of adipogenic/lipogenic genes such as peroxisome proliferator activated receptor gamma (PPARγ), sterol regulatory element binding transcription factor 1 (Srebf1), adipogenin (Adig), and fatty acid binding protein 4 (Fabp4). Further, silencing of Agt gene significantly lowered expression of pro-inflammatory adipokines including interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and monocyte chemotactic protein-1 (MCP-1). In conclusion, this study directly demonstrates critical effects of Agt in adipocyte metabolism and inflammation and further support a potential role for adipose Agt in the pathogenesis of obesity-associated metabolic alterations.
 
| Variations in umbilical cord blood steroid concentrations according to mode of assay. Steroid concentrations in cord blood are displayed as mean ± standard deviation or median and interquartile range, depending on the data available. (A) 17β-estradiol concentrations in cord blood (males and females combined) are displayed from 15 studies. The first 11 utilized immunoassay (predominantly RIA); the remaining 4 used LC-MS/MS, apart from the studies by Hill et al. (41), which used GC-MS. (B) Testosterone concentrations in cord blood (males or mixed
Variations in umbilical cord blood steroid concentrations according to mode of assay. Steroid concentrations in cord blood are displayed as mean ± standard deviation or median and interquartile range, depending on the data available. (A) 17β-estradiol concentrations in cord blood (males and females combined) are displayed from 15 studies. The first 11 utilized immunoassay (predominantly RIA); the remaining 4 used LC–MS/MS, apart from the studies by Hill et al. (41), which used GC–MS. (B) Testosterone concentrations in cord blood (males or mixed male/female samples). Of the 17 studies included, the final 5 employed mass spectrometry. The last study displayed on each chart has the largest samples size (n = 860) and extensive assay validation for cord blood. The steroid values from the studies by Hill et al. (41) are the mean of the published umbilical artery and vein values. The figure demonstrates the influence of assay characteristics on sex steroid values, although the lack of assay specificity is much more significant for measurement of testosterone compared to estradiol.
Summary of the studies examining the relationship between cord hormone concentrations and human development.
Accurately measuring hormone exposure during prenatal life presents a methodological challenge and there is currently no "gold standard" approach. Ideally, circulating fetal hormone levels would be measured at repeated time points during pregnancy. However, it is not currently possible to obtain fetal blood samples without significant risk to the fetus, and therefore surrogate markers of fetal hormone levels must be utilized. Umbilical cord blood can be readily obtained at birth and largely reflects fetal circulation in late gestation. This review examines the accuracy and biological interpretation of the measurement of androgens and estrogens in cord blood. The use of cord blood hormones to understand and investigate human development is then discussed.
 
Objective: Congenital hyperinsulinism (CHI) is a rare condition of hypoglycemia where therapeutic options are limited and often complicated by side-effects. Omega-3-polyunsaturated fatty acids (PUFA), which can suppress cardiac myocyte electrical activity, may also reduce ion channel activity in insulin-secreting cells. PUFA supplements in combination with standard medical treatment may improve glucose profile and may reduce glycemic variability in diazoxide-responsive CHI. Design: Open label pilot trial with MaxEPAR liquid (eicosapentaenoic and docosahexaenoic acid) PUFA (3 ml/day for 21 days) in diazoxide-responsive CHI patients (https://eudract.ema.europa.eu/, EudraCT number 201100363333). Methods: Glucose levels were monitored pre-treatment, end of treatment, and at follow-up by subcutaneous continuous glucose monitoring systems (CGMS) in 13 patients (7 girls) who received PUFA. Outcome measures were an improved glucose profile, reduced glycemic variability quantified by a reduction in the frequency of glucose levels <4 and >10 mmol/l, and safety of PUFA. All children were analyzed either as intention to treat (n = 13) or as per protocol (n = 7). Results: Mean (%) CGMS glucose levels increased by 0.1 mmol/l (2%) in intention to treat and by 0.4 mmol/l (8%) in per protocol analysis (n = 7). The frequency of CGMS <4 mmol/l was significantly less at the end of treatment than in the pre-treatment period [556 (7%) vs. 749 (10%)]. Similarly, the frequency of CGMS >10 mmol/l, was also less at the end of treatment [27 (0.3%) vs. 49 (0.7%)]. Except for one child with increased LDL cholesterol, all safety parameters were normal. Conclusion: MaxEPAR was safe and reduced glycemic variability, but did not increase glucose profiles significantly in diazoxide-responsive CHI. The supplemental value of PUFA should be evaluated in a comprehensive clinical trial.
 
Ghrelin is a peptide hormone that is acylated with a fatty acid, usually n-octanoic acid, at the third amino acid (aa) residue (usually a serine or threonine), and this acylation is known to be essential for ghrelin activity not only in mammals but also in non-mammals, such as fish. However, the modification mechanisms of ghrelin modification in fish are not known. In this study, we elucidated the structure of ghrelin in a teleost, the barfin flounder (Verasper moseri), and determined whether ingested free fatty acids of various chain lengths participated in ghrelin acylation. Complementary DNA cloning revealed the barfin flounder prepro-ghrelin to be a 106-aa peptide and the mature ghrelin to be a 20-aa peptide (GSSFLSPSHKPPNKGKPPRA). However, purification of ghrelin peptides from stomach extracts demonstrated that the major form of the hormone was a 19-aa decanoylated peptide [GSS(C10:0)FLSPSHKPPNKGKPPR] missing the last alanine of the 20-aa peptide. Ingestion of feed enriched with n-heptanoic acid (C7), n-octanoic acid (C8), or n-non-anoic acid (C9) changed the modification status of the peptide: ingestion of C8 or C9 increased the amount of C8:0 or C9:0 19-aa ghrelin, respectively, but no C7:0 ghrelin was isolated after ingestion of C7. These results indicate that ingested free fatty acids are substrates for ghrelin acylation in the barfin flounder, but the types of free fatty acids utilized as substrates may be limited.
 
A) Tracing studies with tritium-labeled palmitic acid reveal that MUFAs such as oleic acid slightly reduce the total amount of [3H]palmitic acid containing DAG + TG in the cellular lipid fraction. In addition, oleic acid leads to a preferential incorporation of [3H]palmitic acid into TG vs. DAG. (B) Total amount of tritium-labeled metabolites recovered in the total cellular lipid fraction was decreased in the presence of oleic acid. Adapted from Sieber et al. (37). n = 9; *p < 0.01, **p < 0.00001.
Working model for increased plasma FFA levels and/or a shift toward SFAs on podocytes. In obesity and type 2 diabetes, increased adipose tissue lipolysis and/or a FFA uptake defect of adipocytes together with increased dietary FFA intake results in elevated plasma FFAs and a “spillover” of FFAs to non-adipose tissues including the kidney and podocytes. Podocytes may adapt to the altered lipid environment by upregulating fatty acid β-oxidation, TG synthesis, and the adaptive branch of the UPR. However, impaired adaptive capacity (e.g., genetic) or chronic “overload” leading to accumulation of toxic FFA metabolites and/or excessive TG storage may lead to diminished podocyte function and ultimately podocyte death resulting in obesity-related glomerulopathy and DN.
Podocyte injury and loss critically contribute to the pathogenesis of proteinuric kidney diseases including diabetic nephropathy. Deregulated lipid metabolism with disturbed free fatty acid (FFA) metabolism is a characteristic of metabolically unhealthy obesity and type 2 diabetes and likely contributes to end-stage kidney disease irrespective of the underlying kidney disease. In the current review, we summarize recent findings related to FFAs and altered renal FFA metabolism with a special focus on podocytes. We will outline the opposing effects of saturated and monounsaturated FFAs and a particular emphasis will be given to the underlying molecular mechanisms involving insulin resistance and endoplasmic reticulum homeostasis. Finally, recent data suggesting a critical role of renal FFA metabolism to adapt to an altered lipid environment will be discussed.
 
| Summary of long chain FFA GPCR agonist pharmacology.
Amino acid sequences for human long chain FFA GPCRs. Diagrams represent FFA1 (A), GenBank NM_005303) and the short isoform of GPR120S [(B), BC101175] with the position of the 16 amino acid insert in GPR120L (NM_181745) indicated in the inset. Putative glycosylation sites are indicated on the extracellular Asn residues by white on black, while the conserved disulfide bridge from extracellular loop 2, and two unproven palmitoylation sites in GPR120, are also indicated by black on gray Cys residues. Basic Arg residues (white on red) have been implicated in recognizing the carboxylate anions of FFA and other agonists (Sum et al., 2007; Suzuki et al., 2008). A number of other residues have been identified as important for GW9508 binding to FFA1, some of which are indicated here (black on blue; Sum et al., 2007); however the specificity with which these mutations alter GW9508 binding, compared to more general effects on FFA1 activation has since been questioned (Smith et al., 2009).
Chemical structures of example FFA GPCR agonists (A) and three reported FFA1 antagonists (B). Information on agonist pharmacology, with references, is provided in Table 1. The structure of Metabolex example 36 is reproduced from the relevant GPR120 agonist patent (Ma et al., 2010) and two Banyu compounds are shown from isoindolin-1-one derivativesa (cpd 2; Arakawa et al., 2010) and the phenyl-isoxazol-3-ol seriesb (cpd 15; Hashimoto et al., 2010). Both GW1100 (Briscoe et al., 2006), Pfizer compound 15i (Humphries et al., 2009), and DC260126 (Hu et al., 2009) inhibited agonist stimulated FFA1 receptor calcium responses in transfected cells with respective pIC50 values of 6.0, 7.7, and 6.0.
Summary of functional assays available to investigate FFA G protein coupled receptor pharmacology in cell lines. The indirect approaches to assessing ligand binding indicated here are discussed further in Section “Determining Ligand Affinity at FFA1 and GPR120.” Several signaling endpoints related to the G protein (FFA1, GPR120) or β-arrestin pathways (GPR120) can be measured using the example assays in italics (see text, Functional Assessment of FFA1 and GPR120 Pharmacology) culminating in assessment of function at the cellular level. Example data from our own laboratory illustrates (i) calcium responses in HEK293 cells expressing human GPR40 and stimulated with the C18:2 FFA linoleic acid, and (ii) internalization of human GPR120S–YFP receptors (green), following vehicle or oleic acid treatment, also in transfected HEK293 cells. In the image panels nuclei are counterstained with the dye H33342 (blue).
Discovery of G protein coupled receptors for long chain free fatty acids (FFAs), FFA1 (GPR40) and GPR120, has expanded our understanding of these nutrients as signaling molecules. These receptors have emerged as important sensors for FFA levels in the circulation or the gut lumen, based on evidence from in vitro and rodent models, and an increasing number of human studies. Here we consider their promise as therapeutic targets for metabolic disease, including type 2 diabetes and obesity. FFA1 directly mediates acute FFA-induced glucose-stimulated insulin secretion in pancreatic beta-cells, while GPR120 and FFA1 trigger release of incretins from intestinal endocrine cells, and so indirectly enhance insulin secretion and promote satiety. GPR120 signaling in adipocytes and macrophages also results in insulin sensitizing and beneficial anti-inflammatory effects. Drug discovery has focused on agonists to replicate acute benefits of FFA receptor signaling, with promising early results for FFA1 agonists in man. Controversy surrounding chronic effects of FFA1 on beta-cells illustrates that long term benefits of antagonists also need exploring. It has proved challenging to generate highly selective potent ligands for FFA1 or GPR120 subtypes, given that both receptors have hydrophobic orthosteric binding sites, which are not completely defined and have modest ligand affinity. Structure activity relationships are also reliant on functional read outs, in the absence of robust binding assays to provide direct affinity estimates. Nevertheless synthetic ligands have already helped dissect specific contributions of FFA1 and GPR120 signaling from the many possible cellular effects of FFAs. Approaches including use of fluorescent ligand binding assays, and targeting allosteric receptor sites, may improve further pre-clinical ligand development at these receptors, to exploit their unique potential to target multiple facets of diabetes.
 
Estrogen rapidly modulates hippocampal synaptic plasticity by activating selective membrane-associated receptors. Reorganization of the actin cytoskeleton and stimulation of mammalian target of rapamycin (mTOR)-mediated protein synthesis are two major events required for the consolidation of hippocampal long-term potentiation and memory. Estradiol regulates synaptic plasticity by interacting with both processes, but the underlying molecular mechanisms are not yet fully understood. Here, we used acute rat hippocampal slices to analyze the mechanisms underlying rapid changes in mTOR activity and actin polymerization elicited by estradiol. Estradiol-induced mTOR phosphorylation was preceded by rapid and transient activation of both extracellular signal-regulated kinase (ERK) and protein kinase B (Akt) and by phosphatase and tensin homolog (PTEN) degradation. These effects were prevented by calpain and ERK inhibitors. Estradiol-induced mTOR stimulation did not require activation of classical estrogen receptors (ER), as specific ERα and ERβ agonists (PPT and DPN, respectively) failed to mimic this effect, and ER antagonists could not block it. Estradiol rapidly activated both RhoA and p21-activated kinase (PAK). Furthermore, a specific inhibitor of RhoA kinase (ROCK), H1152, and a potent and specific PAK inhibitor, PF-3758309, blocked estradiol-induced cofilin phosphorylation and actin polymerization. ER antagonists also blocked these effects of estrogen. Consistently, both PPT and DPN stimulated PAK and cofilin phosphorylation as well as actin polymerization. Finally, the effects of estradiol on actin polymerization were insensitive to protein synthesis inhibitors, but its stimulation of mTOR activity was impaired by latrunculin A, a drug that disrupts actin filaments. Taken together, our results indicate that estradiol regulates local protein synthesis and cytoskeletal reorganization via different molecular mechanisms and signaling pathways.
 
Schematic representation of the steps involved in transcription, translation, post-translational modification, oligomerization, and secretion of adiponectin. Several transcription factors (top left) which mediate adiponectin gene transcription are regulated to increase (thiazolidinedione, TZD) or decrease (tumor necrosis factor-alpha, TNF-α) adiponectin expression. Monomeric adiponectin (mAd) is posttranslationally modified and further oligomerized to form trimers (low molecular weight, LMW), hexamers (medium, MMW) and oligomeric (high, HMW) forms. Various mechanisms (bottom right) mediate this oligomerization and secretion resulting in secretion of HMW, MMW, and LMW forms.
Endocrine and autocrine/paracrine effects of adiponectin. The figure indicates that adiponectin in circulation (blood vessel, center) is derived primarily from adipose tissue (top). Circulating adiponectin can travel to numerous tissues and mediate endocrine effects. In addition, several tissues can also produce adiponectin (solid gray arrow) which can then act locally (twisted gray arrow) to mediate functional autocrine or paracrine effect.
The widespread physiological actions of adiponectin have now been well characterized as clinical studies and works in animal models have established strong correlations between circulating adiponectin level and various disease-related outcomes. Thus, conventional thinking attributes many of adiponectin's beneficial effects to endocrine actions of adipose-derived adiponectin. However, it is now clear that several tissues can themselves produce adiponectin and there is growing evidence that locally produced adiponectin can mediate functionally important autocrine or paracrine effects. In this review article we discuss regulation of adiponectin production, its mechanism of action via receptor isoforms and signaling pathways, and its principal physiological effects (i.e., metabolic and cardiovascular). The role of endocrine actions of adiponectin and changes in local production of adiponectin or its receptors in whole body physiology is discussed.
 
In the zebra finch brain, several forebrain areas contain dense accumulations of aromatase-expressing neurons (A). At higher magnification (B), a substantive portion of the aromatase immunoreactivity is visible in fine fibers (asterisks) that extend a considerable distance from the cell body itself. Sometimes, as in HVC (C) puncta (asterisks) visible as brown deposits following aromatase ICC are clearly visible in an otherwise unstained nucleus (blue soma are Nissl stained cells).
| Criteria that define neuromodulation as applied to synaptocrine signaling.
Synaptocrine signaling: androgenic precursors (orange hexagons) from the extracellular space, adjacent cells such as astroglia, or within individual neurons are available as substrates for the enzyme aromatase (red triangles) located within presynaptic boutons. Estrogens (green hexagons) synthesized within the presynaptic bouton are then available to bind presynaptic ERs (gray ovals) and modulate the dynamics of presynaptic neurotransmitter release. Synaptocrine estrogens may also diffuse into the synaptic cleft and thereby interact with post-synaptic ion channel receptors (brown channel), and/or post-synaptic ERs (gray ovals, cytosolic) or membrane ERs (mER; gray ovals, membrane-bound). Synaptocrine estrogens may therefore modulate post-synaptic neurotransmitter receptors and/or have genomic effects on the post-synaptic cell. These scenarios are most likely to occur when the cell membrane is at rest (i.e., not depolarized) and when voltage-gated calcium channels (VGCCs) are closed. In contrast, (right panel), depolarization of the synaptic neuron and concomitant Ca2+ influx into the presynaptic bouton may activate a calcium-dependent kinase (kin), phosphorylate presynaptic aromatase (P), and thereby decrease synaptocrine estrogen synthesis, while simultaneously potentially increasing the local concentration of androgens.
Classically, the modulation of brain function and behavior by steroid hormones was linked exclusively to secretion by peripheral endocrine glands. Subsequently, steroid actions within the brain were shown dependent upon either synthesis and secretion by peripheral organs or by production within the CNS itself using peripheral sources of precursors. Discovery of the estrogen-synthetic enzyme aromatase in brain further bolstered the latter view and served as a catalyst for expanding concepts of neurosteroidogenesis. In parallel research, several steroids, including estradiol, were found to have rapid effects on neuronal excitability, partially explained by novel actions at neuronal membranes. Recent findings from multiple levels of analysis and labs necessitate an updated view on how steroids are delivered to neural circuits. There is now considerable evidence for expression of the aromatase enzyme within synaptic boutons in the vertebrate CNS. Furthermore, additional work now directly couples rapid regulation of neuroestrogen synthesis with neurophysiological and behavioral outcomes. In this review we summarize evidence for targeted and acute synaptic estrogen synthesis and perisynaptic estrogen actions in the CNS of songbirds. We evaluate these findings in the context of criteria associated with classic neuromodulatory signaling. We term this novel form of signaling "synaptocrine," and discuss its implications.
 
7α-Hydroxypregnenolone synthesis by cytochrome P4507α in the brain of newts and quail. See the text for details.
Schematic model depicting the action of 7α-hydroxypregnenolone on the regulation of locomotor activity in male newt. 7α-Hydroxypregnenolone synthesized actively in the diencephalon and rhombencephalon, by acting on dopaminergic neurons localized in the PT and VTA, may induce dopamine release from their terminals in rostral brain regions, notably in the striatum and nucleus accumbens (NA), and consequently increase locomotor activity of male newt. See the text for details.
Schematic model depicting the action of melatonin on the regulation of diurnal changes in 7α-hydroxypregnenolone synthesis and locomotor activity in quail. Melatonin acts to reduce cytochrome P4507α expression through melatonin receptor-mediated mechanisms. Melatonin derived from the pineal gland and eyes regulates 7α-hydroxypregnenolone synthesis in the brain, thus inducing diurnal locomotor changes. See the text for details.
Schematic model depicting the action of PRL on the regulation of seasonal changes in 7α-hydroxypregnenolone synthesis and locomotor activity in newts. PRL synthesized in the adenohypophysis, by acting on Mg neurons in the hypothalamus, induces the expression of cytochrome P4507α. Cytochrome P4507α and PRL receptor (PRLR) are colocalized in Mg neurons. Thus, prolactin, an adenohypophyseal hormone, regulates 7α-hydroxypregnenolone synthesis in the brain, and also induces seasonal locomotor changes. See the text for details.
Previous studies over the past two decades have demonstrated that the brain and other nervous systems possess key steroidogenic enzymes and produces pregnenolone and other various neurosteroids in vertebrates in general. Recently, 7α-hydroxypregnenolone, a novel bioactive neurosteroid, was identified in the brain of newts and quail. Importantly, this novel neurosteroid is produced from pregnenolone through the enzymatic activity of cytochrome P450(7α) and acts on brain tissue as a neuronal modulator to stimulate locomotor activity in these vertebrates. Subsequently, the mode of action of 7α-hydroxypregnenolone was demonstrated. 7α-Hydroxypregnenolone stimulates locomotor activity through activation of the dopaminergic system. To understand the functional significance of 7α-hydroxypregnenolone in the regulation of locomotor activity, diurnal, and seasonal changes in 7α-hydroxypregnenolone synthesis were further characterized. Melatonin derived from the pineal gland and eyes regulates 7α-hydroxypregnenolone synthesis in the brain, thus inducing diurnal locomotor changes. Prolactin, an adenohypophyseal hormone, regulates 7α-hydroxypregnenolone synthesis in the brain, and also induces seasonal locomotor changes. In addition, 7α-hydroxypregnenolone mediates corticosterone action to modulate locomotor activity under stress. This review summarizes the current knowledge regarding the mode of action and functional significance of 7α-hydroxypregnenolone, a newly identified bioactive neurosteroid stimulating locomotor activity.
 
The figure depicts the differential brain accessibility of a high or a low dose of peripherally administered ghrelin and the potential mechanisms mediating ghrelin brain accessibility. In our study, a set of mice were subcutaneously injected with a high (0.6 nmol/g BW) or a low (0.06 nmol/g BW) dose of fluorescent ghrelin and perfused 15 min later. Brains were removed, post-fixed, cryoprotected, and coronally cut. The neuroanatomical mapping of the presence of the ghrelin tracer was performed by the analysis of the amplified fluorescein-immunoreactive signal achieved by an anti-fluorescein antibody followed by biotinylated secondary antibody, streptavidin–peroxidase, and a chromogenic reaction. Stained brain sections were mounted and bright-field images were acquired with a digital camera at 16 bit/pixel. Finally, pixel intensity values were converted into optical density data by taking the negative decimal logarithm of the original value divided by the white flat field value and pseudocolored. Yellow to blue coloring represents higher to lower levels of fluorescein-immunoreactive signal. (A) displays a schematic illustration representing the distribution of fluorescein signal in a mouse brain after the peripheral administration of the low (left) or the high (right) dose of the ghrelin tracer. AP, area postrema; ARC, arcuate nucleus; ME, median eminence; PVR, periventricular regions; 3V, third ventricle; 4V, fourth ventricle. (B,C) show the actual images of mouse coronal brain sections, at the rostro-caudal levels of the brain labeled with dashed lines in the (A). Letters a, b, c, and d label corresponding brain levels and ghrelin doses between (A) and (B,C). Insets in each microphotography show higher magnification images. Scale bars: 500 μm in low magnification, 100 μm in high magnification. (D) displays a schematic diagram of the potential mechanisms mediating the ghrelin brain accessibility. CVOs, circumventricular organs; CSF, cerebrospinal fluid.
The nervous and endocrine systems act together to regulate all physiological processes essential for the body homeostasis control. Given the strict communication restrictions that the brain-blood barrier (BBB) imposes, the interplay between these two systems requires a variety of delicate anatomical interfaces and physiological mechanisms that guarantee the precise function of the neuroendocrine system as a whole. The study of the mechanisms by which hormones act in the brain in order to regulate specific neuronal populations is a research topic rather neglected. Our group studies the neuronal circuitries and molecular mechanisms by which the stomach-produced hormone ghrelin regulates appetite and other physiological functions. A clear notion of the brain targets of peripheral ghrelin is essential for the comprehensive understanding of the physiological role of this hormone. Ghrelin is called "the hunger hormone" since it is the only known orexigenic peptide hormone. The target for ghrelin orexigenic actions is the brain, which contains a variety of ghrelin-responsive nuclei; however, several evidences suggest that the accessibility of peripheral ghrelin to the brain is strikingly low. Here, we briefly summarize the current knowledge in this topic and discuss this intriguing neuroendocrinological issue.
 
Neurosteroid formation in the Purkinje cell during development. The Purkinje cell possesses several kinds of steroidogenic enzymes. The expression of P450scc remains during neonatal development and in adulthood, indicating the constant production of pregnenolone. This neuron also produces actively progesterone due to an increase of 3β-HSD activity only during neonatal life. Allopregnanolone (3α,5α-tetrahydroprogesterone) is also metabolized by the enzymes 5α-reductase and 3α-HSD from progesterone during neonatal life. Estrogen formation in the Purkinje cell also occurs in the neonate because this neuron further expresses P45017α,lyase and P450arom. See the text for details. StAR, steroidogenic acute regulatory protein; P450scc, cytochrome P450 side-chain cleavage enzyme; 3β-HSD, 3β-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase; P45017α,lyase, cytochrome P450 17α-hydroxylase/c17,20-lyase; 17β-HSD, 17β-hydroxysteroid dehydrogenase; 3α-HSD, 3α-hydroxysteroid dehydrogenase; P450arom, cytochrome P450 aromatase.
Schematic model depicting possible actions of progesterone in the Purkinje cell during development. Progesterone acts on the Purkinje cell through intranuclear receptor (PR)-mediated mechanisms that promote dendritic growth, spinogenesis, and synaptogenesis in this neuron by genomic mechanisms. Thus, progesterone produced in the Purkinje cell may mediate its actions through an “intracrine” mechanism. Progesterone may induce the expression of some neurotrophic factors that directly promote Purkinje dendritic growth, spinogenesis, and synaptogenesis during neonatal life. Progesterone may also act on Purkinje cells through the mechanisms mediated by 25-Dx, which is associated with membrane structures of the endoplasmic reticulum and Golgi apparatus. See the text for details. StAR, steroidogenic acute regulatory protein; P450scc, cytochrome P450 side-chain cleavage enzyme; 3β-HSD, 3β-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase; PR, progesterone receptor; PRE, progesterone response element; Golgi, Golgi apparatus; ER, endoplasmic reticulum.
Schematic model depicting possible actions of estradiol in the Purkinje cell during development. Estradiol acts on the Purkinje cell through intranuclear receptor (ERβ)-mediated mechanisms that promote dendritic growth, spinogenesis, and synaptogenesis in this neuron by genomic mechanisms. Both Purkinje cells and granule cells express BDNF and TrkB, a receptor for BDNF. Estradiol induces the expression of BDNF, which may act on Purkinje cells and granule cells through TrkB-mediated mechanisms to promote Purkinje dendritic growth, spinogenesis, and synaptogenesis. See the text for details. StAR, steroidogenic acute regulatory protein; P450scc, cytochrome P450 side-chain cleavage enzyme; 3β-HSD, 3β-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase; P45017α,lyase, cytochrome P450 17α-hydroxylase/c17,20-lyase; 17β-HSD, 17β-hydroxysteroid dehydrogenase; P450arom, cytochrome P450 aromatase; ERβ, estrogen receptor-β; ERE, estrogen response element; BDNF, brain-derived neurotrophic factor; TrkB, BDNF receptor.
Summary of possible actions of neurosteroids in the Purkinje cell during development. The Purkinje cell actively synthesizes progesterone, allopregnanolone, and estradiol from cholesterol during neonatal life when cerebellar neuronal circuit formation occurs in mammals. Progesterone acts on the Purkinje cell through intranuclear receptor (PR)-mediated mechanisms that promote dendritic growth, spinogenesis, and synaptogenesis in this neuron by genomic mechanisms. Progesterone may also promote dendritic growth, spinogenesis, and synaptogenesis via 25-Dx as well as its nuclear receptor in the Purkinje cell in the neonate. Estradiol acts on the Purkinje cell through intranuclear receptor (ER)-mediated mechanisms that promote dendritic growth, spinogenesis, and synaptogenesis in this neuron by genomic mechanisms. Allopregnanolone is involved in Purkinje cell survival. See the text for details. PR, progesterone receptor; ER, estrogen receptor.
The brain has traditionally been considered to be a target site of peripheral steroid hormones. In addition to this classical concept, we now know that the brain has the capacity to synthesize steroids de novo from cholesterol, the so-called "neurosteroids." In the middle 1990s, the Purkinje cell, an important cerebellar neuron, was identified as a major site for neurosteroid formation in the brain of mammals and other vertebrates. This discovery has provided the opportunity to understand neuronal neurosteroidogenesis in the brain. In addition, biological actions of neurosteroids are becoming clear by the studies using the Purkinje cell, an excellent cellular model, which is known to play an important role in memory and learning processes. Based on the studies on mammals over the past decade, it is considered that the Purkinje cell actively synthesizes progesterone and estradiol from cholesterol during neonatal life, when cerebellar neuronal circuit formation occurs. Both progesterone and estradiol promote dendritic growth, spinogenesis, and synaptogenesis via each cognate nuclear receptor in the developing Purkinje cell. Such neurosteroid actions mediated by neurotrophic factors may contribute to the formation of cerebellar neuronal circuit during neonatal life. 3α,5α-Tetrahydroprogesterone (allopregnanolone), a progesterone metabolite, is also synthesized in the cerebellum and considered to act as a survival factor of Purkinje cells in the neonate. This review summarizes the current knowledge regarding the biosynthesis, mode of action, and functional significance of neurosteroids in the Purkinje cell during development in terms of synaptic formation of cerebellar neuronal networks.
 
Pathways for estrogen to convert tumor promoting effects in cells in the fallopian tubes and the ovaries. (a.) Activation of the nuclear estrogen receptor-α (ER-α) leads to the transcriptional activation of estrogen-responsive genes, which stimulate cell proliferation. (b.) Binding to membrane-bound G-protein-coupled estrogen receptor (GPER) activates second messenger systems. In cancer cells, estrogen induces extracellular-signal regulated kinase (ERK), phosphoinositide 3-kinase (PI3K), and epidermal growth factor receptor (EGFR) leading to enhanced cell proliferation. (c.) The formation of reactive metabolites leads to the generation of mutagenic DNA adducts. Free radicals from the metabolic activation of estrogens will cause mutations. Accumulation of mutations will lead to neoplastic transformation of proliferating cells (10, 42, 44–46)
| Origins and significant mutations of the EOC subtypes.
Pathway for 17β-estradiol formation in cancer cells. Estrone-sulfate (E1-S), a precursor for the most active estrogen 17β-estradiol (E2), androstenediol (5-Diol-S), and dehydroepiandrosterone sulfate (DHEA-S) are taken up from the blood into cancer cells by transporters from the organic anion transporting polypeptide family (OATPs) and other members of the solute carriers (SLCs). In the sulfatase pathway, E1-S is converted through steroid sulfatase (STS) to estrone (E1), which is transformed via the reductive 17β-hydroxysteroid dehydrogenases (17β-HSD red) to E2. E2 as the most active estrogen binds and activates estrogen receptors (ERs). In the reverse pathway from E2 to E1-S, the oxidative 17β-hydroxysteroid dehydrogenases (17β-HSDox) convert E2 to E1. The estrogen sulfotransferase SULT1E1 inactivates estrogens by adding sulfate to hydroxyl-groups on the steroid ring. In the aromatase pathway, E2 is produced from testosterone by the aromatase. Testosterone itself is formed from 5-androstenediol (5-Diol) via 3β-HSD. 5-Diol is generated by the removal of the sulfate from 5-Diol-S via STS. Also 5-Diol can activate ER, but with lower affinity than E2. In an alternative way, testosterone is derived from the conversion of DHEA-S to androstenedione (4-Dione) via DHEA. Finally, 17β-HSD transforms 4-Dione to testosterone (55).
Ovarian cancer is still the deadliest of all gynecologic malignancies in women worldwide. This is attributed to two main features of these tumors, namely, (i) a diagnosis at an advanced tumor stage, and, (ii) the rapid onset of resistance to standard chemotherapy after an initial successful therapy with platin- and taxol-derivatives. Therefore, novel targets for an early diagnosis and better treatment options for these tumors are urgently needed. Epidemiological data show that induction and biology of ovarian cancer is related to life-time estrogen exposure. Also experimental data reveal that ovarian cancer cells share a number of estrogen regulated pathways with other hormone-dependent cancers, e.g., breast and endometrial cancer. However, ovarian cancer is a heterogeneous disease and the subtypes are quite different with respect to mutations, origins, behaviors, markers, and prognosis and respond differently to standard chemotherapy. Therefore, a characterization of ovarian cancer subtypes may lead to better treatment options for the various subtypes and in particular for the most frequently observed high-grade serous ovarian carcinoma. For this intention, further studies on estrogen-related pathways and estrogen formation in ovarian cancer cells are warranted. The review gives an overview on ovarian cancer subtypes and explains the role of estrogen in ovarian cancer. Furthermore, enzymes active to synthesize and metabolize estrogens are described and strategies to target these pathways are discussed.
 
Estradiol plays a pivotal role in the control of gonadotropin-releasing hormone (GnRH) neuronal function and female reproduction. While positive and negative feedback actions of estradiol that enhance and suppress release of GnRH and LH are primarily mediated through estrogen receptor alpha located in interneurons, a series of recent studies in our laboratory indicate that rapid excitatory actions of estradiol also directly modify GnRH neuronal activity. We observed this phenomenon in cultured primate GnRH neurons, but similar rapid direct actions of estradiol are also described in cultured GnRH neurons and green fluorescent protein-labeled GnRH neurons of mice. Importantly, rapid direct action of estradiol in GnRH neurons is mediated through membrane or membrane associated receptors, such as GPR30, STX-sensitive receptors, and ERβ. In this review, possible implications of this rapid estradiol action in GnRH neurons are discussed.
 
The alignment of GnIH (RFRP) precursor polypeptides of birds and mammals. The amino acid sequence of GnIH, GnIH-RP-1, GnIH-RP-2, RFRP-1, RFRP-3 with Gly (G) as an amidation signal and Arg (R) or Lys (K) as an endoproteolytic basic amino acid at the C-termini are shown in bold. Identified mature peptides are underlined. Endoproteolytic basic amino acid (R or K) at the N-termini are also shown in bold. A broken line indicates the putative human RFRP-2 sequence. Accession numbers are human (NM_022150), macaque (NM_001130827), bovine (NM_174168), rat (NM_023952), Siberian hamster (JF727837), white-crowned sparrow (AB128164), zebra finch (AB522971), European starling (EF486798) and Japanese quail (AB039815).
Two-dimensional representation of the receptor (GPR147) for human GnIH (RFRP). The transmembrane region was predicted using SOSUI (Hirokawa et al., 1998). Glycosylation and disulfide bridge sites were predicted by GPCRDB (Horn et al., 1998). The accession number is AB040104. Adapted from Ubuka et al. (2008b).
Schematic model of GnIH (RFRP) action in the brain and pituitary. GnIH (RFRP) neurons in the brain project their axons to GnRH-I neurons as well as to the median eminence (ME). GnIH receptor (GnIH-R; GPR147) is expressed on GnRH-I neurons as well as gonadotropes. GnIH may thus inhibit gonadotropin synthesis and release by inhibiting the activity of GnRH-I neurons as well as directly inhibiting the pituitary gonadotrope. GnIH (RFRP) neurons may also regulate GnRH-I neurons by regulating the activity of kisspeptin (Kiss) neurons that project to GnRH-I neurons. There are also reports showing that GnIH (RFRP) neurons project their axons to GnRH-II, dopamine, pro-opiomelanocortin (POMC), neuropeptide Y, orexin, melanin-concentrating hormone (MCH), corticotrophin-releasing hormone (CRH) and oxytocin neurons in the brain. GnIH (RFRP) inhibits reproductive behaviors of birds and mammals by possibly acting within the brain. The expression of GnIH (RFRP) is regulated by melatonin, stress, and estradiol-17β (E2). Expressions of melatonin receptor (Mel-R), glucocorticoid receptor (GC-R), or estrogen receptor α (ERα) in GnIH (RFRP) neurons were shown in several species. These mechanisms of action of GnIH (RFRP) on gonadotropin secretion or regulatory mechanism of GnIH (RFRP) expression may vary between species, sexes, and developmental stages.
Gonadotropin-inhibitory hormone (GnIH) was first identified in the Japanese quail as a hypothalamic neuropeptide inhibitor of gonadotropin secretion. Subsequent studies have shown that GnIH is present in the brains of birds including songbirds, and mammals including humans. The identified avian and mammalian GnIH peptides universally possess an LPXRFamide (X = L or Q) motif at their C-termini. Mammalian GnIH peptides are also designated as RFamide-related peptides from their structures. The receptor for GnIH is the G protein-coupled receptor 147 (GPR147), which is thought to be coupled to G(αi) protein. Cell bodies of GnIH neurons are located in the paraventricular nucleus (PVN) in birds and the dorsomedial hypothalamic area (DMH) in mammals. GnIH neurons in the PVN or DMH project to the median eminence to control anterior pituitary function. GPR147 is expressed in the gonadotropes and GnIH suppresses synthesis and release of gonadotropins. It was further shown in immortalized mouse gonadotrope cell line (LβT2 cells) that GnIH inhibits gonadotropin-releasing hormone (GnRH) induced gonadotropin subunit gene transcriptions by inhibiting adenylate cyclase/cAMP/PKA-dependent ERK pathway. GnIH neurons also project to GnRH neurons in the preoptic area, and GnRH neurons express GPR147 in birds and mammals. Accordingly, GnIH may inhibit gonadotropin synthesis and release by decreasing the activity of GnRH neurons as well as directly acting on the gonadotropes. GnIH also inhibits reproductive behavior possibly by acting within the brain. GnIH expression is regulated by a nocturnal hormone melatonin and stress in birds and mammals. Accordingly, GnIH may play a role in translating environmental information to inhibit reproductive physiology and behavior of birds and mammals. Finally, GnIH has therapeutic potential in the treatment of reproductive cycle and hormone-dependent diseases, such as precocious puberty, endometriosis, uterine fibroids, and prostatic and breast cancers.
 
Dehydroepiandrosterone (DHEA) is synthesized from cholesterol by activity of P450scc and P450c17, enzymes that we previously characterized in the developing nervous system. We describe the localization of P450c17 in the differentiated field of the ventral spinal cord in different motor neuron subtypes. We show that, during organogenesis, P450c17 activity is regulated along the antero/posterior axis of the spinal cord concomitantly with the gradient of neurogenesis. To examine whether DHEA may modulate this process, we measured proliferation and differentiation of ventral neural precursors in primary and explant cultures. Our results showed that DHEA-induced the expression of class II protein Nkx6.1, motor neuron precursor Olig-2, and definitive motor neuron marker Isl-1/2. DHEA also promoted proliferation of ventrally committed precursors in isolated spinal cord precursor cultures and in whole spinal cord explants. Both the proliferative and inductive effects of DHEA were dependent on sonic hedgehog signaling. The possibilities that the effects observed with DHEA were due to its metabolism into androgens or to activation of NMDA receptors were excluded. These results support the hypothesis that the tight regulation of DHEA biosynthesis may be a biologic clock restricting the period of ventral neuronal-precursor proliferation, thus controlling the number of pre-committed neurons in the developing neural tube.
 
| Effect of native ghrelin and BIM compounds on cumulative food intake and GH secretion measured during the light cycle in mice. Data represent mean ± SEM. (A) Mean cumulative food intake (90 min) after ip injection of native ghrelin (30 nmol), BIM-28131 (30 nmol), BIM-28163 (150 nmol), and native ghrelin (30 nmol) co-administered with BIM-28163 (150 nmol). ANOVA shows an effect of treatment on 90 min food intake: *p < 0.05 BIM-28163 vs. vehicle, **P < 0.01 BIM-28131 vs. vehicle, and native ghrelin, @ P < 0.05
Effect of native ghrelin and BIM compounds on cumulative food intake and GH secretion measured during the light cycle in mice. Data represent mean ± SEM. (A) Mean cumulative food intake (90 min) after ip injection of native ghrelin (30 nmol), BIM-28131 (30 nmol), BIM-28163 (150 nmol), and native ghrelin (30 nmol) co-administered with BIM-28163 (150 nmol). ANOVA shows an effect of treatment on 90 min food intake: *p < 0.05 BIM-28163 vs. vehicle, **P < 0.01 BIM-28131 vs. vehicle, and native ghrelin, @P < 0.05 BIM-28131 vs. native ghrelin + BIM-28163, Fisher PLSD post hoc test. (B) GH secretion measured by tail bleeding 15 min after ip injection of BIM compounds in the same animals. ANOVA shows an effect of treatment on GH levels: **p < 0.01 native ghrelin vs. vehicle, ***P < 0.001 BIM-28131 vs. vehicle, @P < 0.05 native ghrelin vs. native ghrelin + BIM-28163, @@P < 0.01 BIM-28131 vs. native ghrelin + BIM-28163, ##P < 0.01 native ghrelin or BIM-28131 vs. BIM-28163, Fisher PLSD post hoc test.
Effect of native ghrelin and BIM compounds on cumulative food intake measured with the automated feeding station in mice. Mean cumulative food intake (0–7 h) after ip injection of native ghrelin (30 nmol), BIM-28131 (30 nmol), BIM-28163 (150 nmol), and native ghrelin (30 nmol) co-administered with BIM-28163 (150 nmol). Data represent mean ± SEM. Repeated measures ANOVA over time (7 h) and across treatments shows an interaction between time and treatment on cumulative food intake. P = 0.06 BIM-28131 vs. vehicle, Fisher PLSD post-hoc test.
Effect of native ghrelin and BIM compounds on cFos immunoreactivity in mice in the ArcN, AP, NTS, and VMH. (A) Number of cFos immunoreactive nuclei and (B) sum of cFos immunoreactive nuclei along the rostro-caudal extent of the ArcN (2.3–1.6 mm anterior to the interaural line). Data represent mean ± SEM. (A) Repeated measures ANOVA over the rostro-caudal extent of the ArcN shows an effect of treatment on the number of cFos-positive nuclei: P < 0.0001 native ghrelin vs. Vehicle, BIM-28163, and BIM-28131, P < 0.0001 BIM-28131 vs. all other treatments, P < 0.0001 BIM-28163 vs. all other treatments except vehicle, P < 0.01 BIM-28163 vs. vehicle, P < 0.0001 native ghrelin + BIM-28163 vs. vehicle, BIM-28163, and BIM-28131, Fisher PLSD post hoc test. (B) ***P < 0.001 vs. vehicle, #P < 0.05 vs. BIM-28163, ##P < 0.01 vs. BIM-28163, ###P < 0.001 vs. BIM-28163, @@P < 0.01 vs. ghrelin + BIM-28163, Fisher PLSD post hoc test. (C,D) Number of cFos immunoreactive nuclei in the NTS and AP (3.7 mm posterior to the interaural line). Data represent mean ± SEM. ANOVA shows an effect of treatment on the number of cFos nuclei in the NTS and AP. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. vehicle, #P < 0.05 vs. 28163, Fisher PLSD post hoc test. (E) Number of cFos immunoreactive nuclei in the VMH (2.5–2.3 mm anterior to the interaural line). Data represent mean ± SEM. No significant effect of treatments is observed in the VMH. (F) Summary of the effect of the different BIM compounds on cFos activation in the ArcN, NTS, and AP. Cc, central canal.
Effect of native ghrelin and BIM compounds on cFos immunoreactivity in ArcN NPY neurons in NPY-Renilla GFP mice. Representative confocal microphotographs showing GFP neurons (green), cFos nuclei (red), and the merge of both signals (yellow/orange) in coronal sections of the ArcN at approximately 2.1–1.9 mm anterior to the interaural line. Data represent mean ± SEM. Scale bar represents 50 μm in the ArcN. 3V, third ventricle; ME, median eminence.
The stimulatory effects of ghrelin, a 28-AA acylated peptide originally isolated from stomach, on GH secretion and feeding are exclusively mediated through the growth hormone secretagogue 1a receptor (GHS-R1a), the only ghrelin receptor described so far. Several GHS-R1a agonists and antagonists have been developed to treat metabolic or nutritional disorders but their mechanisms of action in the central nervous system remain poorly understood. In the present study, we compared the activity of BIM-28163, a GHS-R1a antagonist and of several agonists, including native ghrelin and the potent synthetic agonist, BIM-28131, to modulate food intake, GH secretion and c-Fos activity in ArcN, NTS and AP in wild-type and NPY-GFP mice. BIM-28131 was as effective as ghrelin in stimulating GH secretion, but more active than ghrelin in inducing feeding. It stimulated cFos activity similarly to ghrelin in the NTS and AP but was more powerful in the ArcN, suggesting that the super-agonist activity of BIM-28131 is mostly mediated in the ArcN. BIM-28163 antagonized ghrelin-induced GH secretion but not ghrelin-induced food consumption and cFos activation, rather it stimulated food intake and cFos activity without affecting GH secretion. The level of cFos activation was dependent on the region considered: BIM-28163 was as active as ghrelin in the NTS, but less active in the ArcN and AP. All compounds also induced cFos immunoreactivity in ArcN NPY neurons but BIM-28131 was the most active. In conclusion, these data demonstrate that two peptide analogs of ghrelin, BIM-28163 and BIM-28131, are powerful stimulators of appetite in mice, acting through pathways and key brain regions involved in the control of appetite that are only partially superimposable from those activated by ghrelin. A better understanding of the molecular pathways activated by these compounds could be useful in devising future therapeutic applications, such as for cachexia and anorexia.
 
CN activation destabilizes the actin cytoskeleton and causes podocyte apoptosis. Phosphorylation of SYN is mediated by PKA and CamKII. Phosphorylated SYN promotes 14–3–3 binding, which protects SYN from degradation by cathepsin L. SYN also binds Rho A, and competitively inhibits binding of Rho A to the ubiquitin ligase Smurf1, which prevents targeting of Rho A for proteasomal degradation. Binding of Rho A to SYN activates Rho A (GTP bound Rho A) and, in turn, induces stress fiber formation and stabilizes the podocyte cytoskeleton. CN dephosphorylates the 14–3–3 docking site in SYN and promotes SYN degradation by cathepsin L. In the absence of SYN, Rho A is targeted for proteosomal degradation, which reduces stress fiber formation and destabilizes the actin cytoskeleton. CN also promotes podocyte apoptosis by dephosphorylation of either NFAT isoforms, Drp1 or BAD. This apoptotic effect is mediated both directly by Drp1- or BAD-dependent activation of mitochondrial apoptotic pathways, as well as indirectly by stimulation of NFAT-dependent gene transcription.
Diseases affecting the glomerulus are the most common cause of end-stage kidney disease in developed countries (1). These disorders are characterized by significant proteinuria, and the level of proteinuria is an independent risk factor for disease progression (2). Podocytes are thought to play a key role in the pathogenesis of glomerular diseases (3, 4). The importance of podocytes in glomerular diseases is highlighted by genetic studies, which have identified mutant podocyte proteins that cause familial forms of nephrosis (5). Because podocytes are terminally differentiated cells with little capacity for replication, their ability to compensate for podocyte loss is limited (3). A component of current therapy is, therefore, focused on reducing podocyte injury by decreasing systemic blood pressure (BP) and inhibition of the renin–angiotensin system (2, 6).
 
The gonadotropin-releasing hormone (GnRH) was originally isolated from the mammalian hypothalamus for its role as the primary regulator of reproductive function. Since its discovery, GnRH has also been shown to be located in non-hypothalamic tissues and is known to have diverse functions. Although the regulation of GnRH synthesis and release has been extensively studied, there is additional evidence to suggest that the processing of GnRH to the metabolite GnRH-(1-5) represents another layer of regulation. The focus of this review will be on the current evidence for the action of the pentapeptide metabolite GnRH-(1-5) in regulating cellular migration. We discuss the potential role of GnRH-(1-5) in regulating GnRH neuronal migration during development. Furthermore, we demonstrate these actions are mediated by the activation of a G protein-coupled receptor. Our findings suggest that GnRH-(1-5) may play a developmental function in addition to regulating developing cells.
 
| Leading actions of chosen neurosteroids.
Schematic synthesis of neurosteroids in the CNS.
Neurosteroids were initially defined as steroid hormones locally synthesized within the nervous tissue. Subsequently, they were described as steroid hormone derivatives that devoid hormonal action but still affect neuronal excitability through modulation of ionotropic receptors. Neurosteroids are further subdivided into natural (produced in the brain) and synthetic. Some authors distinguish between hormonal and regular neurosteroids in the group of natural ones. The latter group, including hormone metabolites like allopregnanolone or tetrahydrodeoxycorticosterone, is devoid of hormonal activity. Both hormones and their derivatives share, however, most of the physiological functions. It is usually very difficult to distinguish the effects of hormones and their metabolites. All these substances may influence seizure phenomena and exhibit neuroprotective effects. Neuroprotection offered by steroid hormones may be realized in both genomic and non-genomic mechanisms and involve regulation of the pro- and anti-apoptotic factors expression, intracellular signaling pathways, neurotransmission, oxidative, and inflammatory processes. Since regular neurosteroids show no affinity for steroid receptors, they may act only in a non-genomic mode. Multiple studies have been conducted so far to show efficacy of neurosteroids in the treatment of the central and peripheral nervous system injury, ischemia, neurodegenerative diseases, or seizures. In this review we focused primarily on neurosteroid mechanisms of action and their role in the process of neurodegeneration. Most of the data refers to results obtained in experimental studies. However, it should be realized that knowledge about neuroactive steroids remains still incomplete and requires confirmation in clinical conditions.
 
Since its development, the bioluminescence resonance energy transfer (BRET) approach has been extensively applied to study G protein-coupled receptors (GPCRs) in real-time and in live cells. One of the major aspects of GPCRs investigated in considerable details is their physical coupling to the heterotrimeric G proteins. As a result, new concepts have emerged, but few questions are still a matter of debate illustrating the complexity of GPCR-G protein interactions and coupling. Here, we summarized the recent advances on our understanding of GPCR-G protein coupling based on BRET approaches and supported by other FRET-based studies. We essentially focused on our recent studies in which we addressed the concept of preassembly vs. the agonist-dependent interaction between the protease-activated receptor 1 (PAR1) and its cognate G proteins. We discussed the concept of agonist-induced conformational changes within the preassembled PAR1-G protein complexes as well as the critical question how the multiple coupling of PAR1 with two different G proteins, Gαi1 and Gα12, but also β-arrestin 1, can be regulated.
 
Growth hormone (GH) activates multiple signal pathways via rainbow trout GH receptors (GHR) transfected in Chinese hamster ovary (CHO)-K1 cells. CHO-K1 cells were stably transfected with cDNA encoding rainbow trout GHR1 and GHR2. Cells were incubated with 100 ng/ml salmonid GH for various times; after which, the cells were collected and lysed. Lysates were separated by SDS-PAGE followed by Western immunoblotting using phospho-specific (pERK 1/2, pAkt, pJAK2, pSTAT5) and total (tERK 1/2; tAkt, tJAK2, tSTAT5) antibodies.
Growth hormone receptors (GHR) subtypes differentially activate (A) ERK, (B) Akt, and (C) STAT5. CHO-K1 cells were stably transfected with cDNA encoding rainbow trout GHR1 and GHR2. Cells were incubated with various concentrations of salmonid GH for 10 min (control is 0 ng/ml); after which time, the cells were collected and lysed. Lysates were separated by SDS-PAGE followed by Western immunoblotting using phospho-specific (pERK 1/2, pAkt, pJAK2, pSTAT5) and total (tERK 1/2; tAkt, tJAK2, tSTAT5) antibodies. Data are expressed as percentage of control and are presented as means ± SEM (n = 8) of blots quantified with a digital imaging system; * designates groups that are significantly different from each other (P < 0.05).
Effects of pathway blockade on growth hormone-induced activation of (A) ERK, (B) Akt, and (C) STAT5. CHO-K1 cells were stably transfected with cDNA encoding rainbow trout GHR1 and GHR2. Transfected cells were pretreated with or without specific inhibitors [10 μM of the MEK inhibitor, U0126; 20 μM of the PI3K inhibitor, LY294002 (LY); 25 μM of the Akt inhibitor, 1L6-hydroxyymethyl-chiro-inositol-2-(R)-2-O-methyl-3-O-octadecyl-sn-glycerocarbonate (Carb); 200 μM of the STAT5 inhibitor, N′-((4-oxo-4H-chroen-3-yl)methylene)nicotinohydrazide (Nico); and 50 μM of the JAK2 inhibitor, 1,2,3,4,5,6-hexabromocyclohexane (Hex)] for 2 h, then treated with or without 100 ng/ml growth hormone (GH) for 10 min (control is 0 ng/ml GH); after which time, the cells were collected and lysed. Cell lysates were separated by SDS-PAGE followed by Western immunoblotting using phospho-specific (pERK1/2, pAkt, or pSTAT) and total (tERK1/2, tAkt, or tSTAT) antibodies. Data are expressed as percentage of control and are presented as means ± SEM (n = 8). For a given GHR subtype, groups with different letters are significantly different from each other (P < 0.05); * designates a significant difference (P < 0.05) between subtypes within a given treatment.
Model of differential activation of signaling pathways by growth hormone receptor (GHR) subtypes. JAK2 activation is essential for propagation of signaling from both GHR1 and GHR2 to the ERK, PI3K/Akt, and STAT5 pathways (details of pathway elements are omitted for simplicity). Cross-talk occurs between the ERK and PI3K/Akt pathways, possibly through Akt activation of c-Raf in the ERK pathway.
Previously, we found that the teleost fish, rainbow trout, possesses two growth hormone receptor (GHR) subtypes that display distinct ligand-binding and agonist-induced regulation features. In this study, we used Chinese hamster ovary-K1 cells stably transfected individually with the two trout GHR subtypes, GHR1 and GHR2, to elucidate receptor-effector pathway linkages. Growth hormone (GH) stimulated rapid (5-10 min) phosphorylation of ERK, Akt, JAk2, and STAT5 in both GHR1- and GHR2-expressing cells; however; STAT5 was activated to a greater extent through GHR1 than through GHR2, whereas ERK and Akt were activated to a greater through GHR2 than through GHR1. Although blockade of the ERK pathway had no effect on the activation of Akt, inhibition of PI3K-Akt partially prevented activation of ERK, suggesting cross-talk between the ERK and PI3K-Akt pathways. JAK2 inhibition completely blocked activation of ERK, Akt, and STAT5, suggesting that all of these pathways link to GHR1 and GHR2 via JAK2. These findings establish important receptor-effector pathway linkages and suggest that the GHR subtypes of teleost fish may be functionally distinct.
 
G protein-coupled receptors are well recognized as being able to activate several signaling pathways through the activation of different G proteins as well as other signaling proteins such as β-arrestins. Therefore, understanding how such multiple GPCR-mediated signaling can be integrated constitute an important aspect. Here, we applied bioluminescence resonance energy transfer (BRET) to shed more light on the G protein coupling profile of trypsin receptor, or protease-activated receptor 2 (PAR2), and its interaction with β-arrestin1. Using YFP and Rluc fusion constructs expressed in COS-7 cells, BRET data revealed a pre-assembly of PAR2 with both Gαi1 and Gαo and a rapid and transient activation of these G proteins upon receptor activation. In contrast, no pre-assembly of PAR2 with Gα12 could be detected and their physical association can be measured with a very slow and sustained kinetics similar to that of β-arrestin1 recruitment. These data demonstrate the coupling of PAR2 with Gαi1, Gαo, and Gα12 in COS-7 cells with differences in the kinetics of GPCR-G protein coupling, a parameter that very likely influences the cellular response. Moreover, this further illustrates that pre-assembly or agonist-induced G protein interaction depends on receptor-G protein pairs indicating another level of complexity and regulation of the signaling of GPCR-G protein complexes and its multiplicity.
 
Top-cited authors
Yara Zayed
  • St. Michael's Hospital
Heyam Hayder
  • York University
Maria Theresa E Montales
  • University of Arkansas for Medical Sciences
Alexandria Beebe
  • Arkansas Children's Hospital
Aditya Chada
  • Emory Hospitals