Corticosteroid-binding globulin (CBG) transports glucocorticoids in blood and is a serine protease inhibitor family member. Human CBG has a reactive center loop (RCL) which, when cleaved by neutrophil elastase (NE), disrupts its steroid-binding activity. Measurements of CBG levels are typically based on steroid-binding capacity or immunoassays. Discrepancies in ELISAs using monoclonal antibodies that discriminate between intact vs RCL-cleaved CBG have been interpreted as evidence that CBG with a cleaved RCL and low affinity for cortisol exists in the circulation. We examined the biochemical properties of plasma CBG in samples with discordant ELISA measurements and sought to identify RCL-cleaved CBG in human blood samples. Plasma CBG-binding capacity and ELISA values were consistent in arterial and venous blood draining skeletal muscle, liver and brain, as well as from a tissue (adipose) expected to contain activated neutrophils in obese individuals. Moreover, RCL-cleaved CBG was undetectable in plasma from critically ill patients, irrespective of whether their ELISA measurements were concordant or discordant. We found no evidence of RCL-cleaved CBG in plasma using a heat-dependent polymerization assay, and CBG that resists immunoprecipitation with a monoclonal antibody designed to specifically recognize an intact RCL, bound steroids with a high affinity. In addition, mass spectrometry confirmed the absence of NE-cleaved CBG in plasma in which ELISA values were highly discordant. Human CBG with a NE-cleaved RCL and low affinity for steroids is absent in blood samples, and CBG ELISA discrepancies likely reflect structural differences that alter epitopes recognized by specific monoclonal antibodies.
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Lesley A Hill, Dimitra A Vassiliadi, Ioanna Dimopoulou, Anna J Anderson, Luke D Boyle, Alixe H M Kilgour, Roland H Stimson, Yoan Machado, Christopher M Overall, Brian R Walker, John G Lewis and Geoffrey L Hammond
Hiroharu Mifune, Yuji Tajiri, Yusuke Sakai, Yukie Kawahara, Kento Hara, Takahiro Sato, Yoshihiro Nishi, Akinori Nishi, Ryouichi Mitsuzono, Tatsuyuki Kakuma and Masayasu Kojima
We previously reported that voluntary exercise contributed to the amelioration of abnormal feeding behavior with a concomitant restoration of ghrelin production in a rat model of obesity, suggesting a possible relationship between exercise and appetite-regulating hormones. Ghrelin is known to be involved in the brain reward circuits via dopamine neurons related to motivational properties. We investigated the relevance of ghrelin as an initiator of voluntary exercise as well as feeding behavior. The plasma ghrelin concentration fluctuates throughout the day with its peak at the beginning of the dark period in the wild-type (WT) mice with voluntary exercise. Although predominant increases in wheel running activity were observed accordant to the peak of plasma ghrelin concentration in the WT mice, those were severely attenuated in the ghrelin-knockout (GKO) mice under either ad libitum or time-restricted feeding. A single injection of ghrelin receptor agonist brought about and reproduced a marked enhancement of wheel running activity, in contrast to no effect by the continuous administration of the same drug. Brain dopamine levels (DAs) were enhanced after food consumption in the WT mice under voluntary exercise. Although the acceleration of DAs were apparently blunted in the GKO mice, they were dramatically revived after the administration of ghrelin receptor agonist, suggesting the relevance of ghrelin in the reward circuit under voluntary exercise. These findings emphasize that the surge of ghrelin plays a crucial role in the formation of motivation for the initiation of voluntary exercise possibly related to the central dopamine system.
Saadia Basharat, Jennifer A Parker, Kevin G Murphy, Stephen R Bloom, Julia C Buckingham and Christopher D John
Obesity is a risk factor for sepsis morbidity and mortality, whereas the hypothalamic–pituitary–adrenal (HPA) axis plays a protective role in the body's defence against sepsis. Sepsis induces a profound systemic immune response and cytokines serve as excellent markers for sepsis as they act as mediators of the immune response. Evidence suggests that the adipokine leptin may play a pathogenic role in sepsis. Mouse endotoxaemic models present with elevated leptin levels and exogenously added leptin increased mortality whereas human septic patients have elevated circulating levels of the soluble leptin receptor (Ob-Re). Evidence suggests that leptin can inhibit the regulation of the HPA axis. Thus, leptin may suppress the HPA axis, impairing its protective role in sepsis. We hypothesised that leptin would attenuate the HPA axis response to sepsis. We investigated the direct effects of an i.p. injection of 2 mg/kg leptin on the HPA axis response to intraperitoneally injected 25 μg/kg lipopolysaccharide (LPS) in the male Wistar rat. We found that LPS potently activated the HPA axis, as shown by significantly increased plasma stress hormones, ACTH and corticosterone, and increased plasma interleukin 1β (IL1β) levels, 2 h after administration. Pre-treatment with leptin, 2 h before LPS administration, did not influence the HPA axis response to LPS. In turn, LPS did not affect plasma leptin levels. Our findings suggest that leptin does not influence HPA function or IL1β secretion in a rat model of LPS-induced sepsis, and thus that leptin is unlikely to be involved in the acute-phase endocrine response to bacterial infection in rats.
David M Golding, Daniel J Rees, Jennifer R Davies, Dinko Relkovic, Hannah V Furby, Irina A Guschina, Anna L Hopkins, Jeffrey S Davies, James L Resnick, Anthony R Isles and Timothy Wells
Prader–Willi syndrome (PWS), a neurodevelopmental disorder caused by loss of paternal gene expression from 15q11–q13, is characterised by growth retardation, hyperphagia and obesity. However, as single gene mutation mouse models for this condition display an incomplete spectrum of the PWS phenotype, we have characterised the metabolic impairment in a mouse model for ‘full’ PWS, in which deletion of the imprinting centre (IC) abolishes paternal gene expression from the entire PWS cluster. We show that PWS-ICdel mice displayed postnatal growth retardation, with reduced body weight, hyperghrelinaemia and marked abdominal leanness; proportionate retroperitoneal, epididymal/omental and inguinal white adipose tissue (WAT) weights being reduced by 82%, 84% and 67%, respectively. PWS-ICdel mice also displayed a 48% reduction in proportionate interscapular brown adipose tissue (isBAT) weight with significant ‘beiging’ of abdominal WAT, and a 2°C increase in interscapular surface body temperature. Maintenance of PWS-ICdel mice under thermoneutral conditions (30°C) suppressed the thermogenic activity in PWS-ICdel males, but failed to elevate the abdominal WAT weight, possibly due to a normalisation of caloric intake. Interestingly, PWS-ICdel mice also showed exaggerated food hoarding behaviour with standard and high-fat diets, but despite becoming hyperphagic when switched to a high-fat diet, PWS-ICdel mice failed to gain weight. This evidence indicates that, unlike humans with PWS, loss of paternal gene expression from the PWS cluster in mice results in abdominal leanness. Although reduced subcutaneous insulation may lead to exaggerated heat loss and thermogenesis, abdominal leanness is likely to arise from a reduced lipid storage capacity rather than increased energy utilisation in BAT.
Karen Piper Hanley, Tom Hearn, Andrew Berry, Melanie J Carvell, Ann-Marie Patch, Louise J Williams, Sarah A Sugden, David I Wilson, Sian Ellard and Neil A Hanley
Neurogenin 3 (NGN3) commits pancreatic progenitors to an islet cell fate. We have induced NGN3 expression and identified upregulation of the gene encoding the Ras-associated small molecular mass GTP-binding protein, RAB3B. RAB3B localised to the cytoplasm of human β-cells, both during the foetal period and post natally. Genes encoding alternative RAB3 proteins and RAB27A were unaltered by NGN3 expression and in human adult islets their transcripts were many fold less prevalent than those of RAB3B. The regulation of insulin exocytosis in rodent β-cells and responsiveness to incretins are reliant on Rab family members, notably Rab3a and Rab27a, but not Rab3b. Our results support an important inter-species difference in regulating insulin exocytosis where RAB3B is the most expressed isoform in human islets.
Young Hoon Son, Seok-Jin Lee, Ki-Baek Lee, Jin-Haeng Lee, Eui Man Jeong, Sun Gun Chung, Sang-Chul Park and In-Gyu Kim
Glucocorticoids play a major role in the development of muscle atrophy in various medical conditions, such as cancer, burn injury, and sepsis, by inhibiting insulin signaling. In this study, we report a new pathway in which glucocorticoids reduce the levels of upstream insulin signaling components by downregulating the transcription of the gene encoding caveolin-1 (CAV1), a scaffolding protein present in the caveolar membrane. Treatment with the glucocorticoid dexamethasone (DEX) decreased CAV1 protein and Cav1 mRNA expression, with a concomitant reduction in insulin receptor alpha (IRα) and IR substrate 1 (IRS1) levels in C2C12 myotubes. On the basis of the results of promoter analysis using deletion mutants and site-directed mutagenesis a negative glucocorticoid-response element in the regulatory region of the Cav1 gene was identified, confirming that Cav1 is a glucocorticoid-target gene. Cav1 knockdown using siRNA decreased the protein levels of IRα and IRS1, and overexpression of Cav1 prevented the DEX-induced decrease in IRα and IRS1 proteins, demonstrating a causal role of Cav1 in the inhibition of insulin signaling. Moreover, injection of adenovirus expressing Cav1 into the gastrocnemius muscle of mice prevented DEX-induced atrophy. These results indicate that CAV1 is a critical regulator of muscle homeostasis, linking glucocorticoid signaling to the insulin signaling pathway, thereby providing a novel target for the prevention of glucocorticoid-induced muscle atrophy.
M J F Newson, G R Pope, E M Roberts, S J Lolait and A-M O'Carroll
The neuropeptide apelin is expressed in hypothalamic paraventricular and supraoptic nuclei and mediates its effects via activation of the apelin receptor (APJ). Evidence suggests a role for apelin and APJ in mediating the neuroendocrine response to stress. To understand the physiological role of APJ in regulation of the hypothalamic–pituitary–adrenal (HPA) axis, we measured ACTH and corticosterone (CORT) plasma levels in male and female mice lacking APJ (APJ knockout, APJ KO) and in wild-type controls, in response to a variety of acute stressors. Exposure to mild restraint, systemic injection of lipopolysaccharide (LPS), insulin-induced hypoglycaemia and forced swim (FS) stressors, elevated plasma ACTH and CORT levels in wild-type mice. Acute mild restraint significantly increased plasma ACTH and CORT to a similar level in APJ KO mice as in wild-type mice. However, an intact APJ was required for a conventional ACTH, but not CORT, response to LPS administration in male mice and to insulin-induced hypoglycaemia in male and female mice. In contrast, APJ KO mice displayed an impaired CORT response to acute FS stress, regardless of gender. These data indicate that APJ has a role in regulation of the HPA axis response to some acute stressors and has a gender-specific function in peripheral immune activation of the HPA axis.
Colin P Sibley
Placental dysfunction underlies major obstetric diseases such as pre-eclampsia and fetal growth restriction (FGR). Whilst there has been a little progress in prophylaxis, there are still no treatments for placental dysfunction in normal obstetric practice. However, a combination of increasingly well-described in vitro systems for studying the human placenta, together with the availability of more appropriate animal models of pre-eclampsia and FGR, has facilitated a recent surge in work aimed at repurposing drugs and therapies, developed for other conditions, as treatments for placental dysfunction. This review: (1) highlights potential candidate drug targets in the placenta – effectors of improved uteroplacental blood flow, anti-oxidants, heme oxygenase induction, inhibition of HIF, induction of cholesterol synthesis pathways, increasing insulin-like growth factor II availability; (2) proposes an experimental pathway for taking a potential drug or treatment for placental dysfunction from concept through to early phase clinical trials, utilizing techniques for studying the human placenta in vitro and small animal models, particularly the mouse, for in vivo studies; (3) describes the data underpinning sildenafil citrate and adenovirus expressing vascular endothelial growth as potential treatments for placental dysfunction and summarizes recent research on other potential treatments. The importance of sharing information from such studies even when no effect is found, or there is an adverse outcome, is highlighted. Finally, the use of adenoviral vectors or nanoparticle carriers coated with homing peptides to selectively target drugs to the placenta is highlighted: such delivery systems could improve efficacy and reduce the side effects of treating the dysfunctional placenta.
Peter Kolkhof and Lars Bärfacker
The cDNA of the mineralocorticoid receptor (MR) was cloned 30 years ago, in 1987. At that time, spirolactone, the first generation of synthetic steroid-based MR antagonists (MRAs), which was identified in preclinical in vivo models, had already been in clinical use for 30 years. Subsequent decades of research and development by Searle & Co., Ciba-Geigy, Roussel Uclaf and Schering AG toward identifying a second generation of much more specific steroidal MRAs were all based on the initial 17-spirolactone construct. The salient example is eplerenone, first described in 1987, coincidentally with the cloning of MR cDNA. Its launch on the market in 2003 paralleled intensive drug discovery programs for a new generation of non-steroidal MRAs. Now, 30 years after the cDNA cloning of MR and 60 years of clinical use of steroidal MRAs, novel non-steroidal MRAs such as apararenone, esaxerenone and finerenone are in late-stage clinical trials in patients with heart failure, chronic kidney disease (CKD), hypertension and liver disease. Finerenone has already been studied in over 2000 patients with heart failure plus chronic kidney disease and/or diabetes, and in patients with diabetic kidney disease, in five phase II clinical trials. Here, we reflect on the history of the various generations of MRAs and review characteristics of the most important steroidal and non-steroidal MRAs.