Edward W. Hillhouse

Secretion of adiponectin by human placenta: differential modulation of adiponectin and its receptors by cytokines.

Aims/hypothesisPregnancy, a state of insulin resistance, is associated with elevated levels of cytokines and profound alterations in metabolism. Serum adiponectin, an adipokine with anti-inflammatory and insulin-sensitising properties, has been shown to be lower in patients with gestational diabetes mellitus, a state of greater insulin resistance than normal pregnancies. Hypothesising that the human placenta is a source of adiponectin, we investigated its expression and secretion, and the regulation by cytokines of adiponectin and its receptors.MethodsReal-time RT-PCR, radioimmunoassay, Western blotting, radioligand binding and immunofluorescent analyses were applied to demonstrate the expression, secretion and functionality of placental adiponectin.ResultsAdiponectin gene expression and protein were found in the human term placenta, with expression primarily in the syncytiotrophoblast. RIA of conditioned media from explant experiments revealed that the placenta can secrete adiponectin in vitro. Addition of conditioned media to HEK-293 cells transfected with the gene for adiponectin receptor-1 (ADIPOR1) altered the phosphorylation status of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase, an effect abolished after preabsorption with adiponectin antibody. Cytokines, including TNF-α, IFN-γ, IL-6 and leptin, differentially modulated placental adiponectin receptors as well as adiponectin gene expression and secretion. Interestingly, in placentae from women with gestational diabetes mellitus, we observed significant downregulation of adiponectin mRNA, significant upregulation of ADIPOR1 expression, and a non-significant increase in ADIPOR2 expression.Conclusions/interpretationOur results indicate that the human placenta produces and secretes adiponectin, and that adiponectin and its receptors are differentially regulated by cytokines and their expression altered in women with gestational diabetes mellitus. Collectively, our novel data suggest that adiponectin may play a role in adapting energy metabolism at the materno-fetal interface.

Identification of Nesfatin-1 in Human and Murine Adipose Tissue: A Novel Depot-Specific Adipokine with Increased Levels in Obesity

Nesfatin-1 is a recently identified anorexigenic peptide derived from its precursor protein, nonesterified fatty acid/nucleobindin 2 (NUCB2). Although the hypothalamus is pivotal for the maintenance of energy homeostasis, adipose tissue plays an important role in the integration of metabolic activity and energy balance by communicating with peripheral organs and the brain via adipokines. Currently no data exist on nesfatin-1 expression, regulation, and secretion in adipose tissue. We therefore investigated NUCB2/nesfatin-1 gene and protein expression in human and murine adipose tissue depots. Additionally, the effects of insulin, dexamethasone, and inflammatory cytokines and the impact of food deprivation and obesity on nesfatin-1 expression were studied by quantitative RT-PCR and Western blotting. We present data showing NUCB2 mRNA (P < 0.001), nesfatin-1 intracellular protein (P < 0.001), and secretion (P < 0.01) were significantly higher in sc adipose tissue compared with other depots. Also, nesfatin-1 protein expression was significantly increased in high-fat-fed mice (P < 0.01) and reduced under food deprivation (P < 0.01) compared with controls. Stimulation of sc adipose tissue explants with inflammatory cytokines (TNFalpha and IL-6), insulin, and dexamethasone resulted in a marked increase in intracellular nesfatin-1 levels. Furthermore, we present evidence that the secretion of nesfatin-1 into the culture media was dramatically increased during the differentiation of 3T3-L1 preadipocytes into adipocytes (P < 0.001) and after treatments with TNF-alpha, IL-6, insulin, and dexamethasone (P < 0.01). In addition, circulating nesfatin-1 levels were higher in high-fat-fed mice (P < 0.05) and showed positive correlation with body mass index in human. We report that nesfatin-1 is a novel depot specific adipokine preferentially produced by sc tissue, with obesity- and food deprivation-regulated expression.

Role of corticotropin-releasing hormone in onset of labour

Corticotropin-releasing hormone (CRH) derived from the placenta is secreted into the maternal circulation in large amounts during the third trimester of human pregnancy and may have an important role in the onset of labour. Although the biological role of CRH remains enigmatic, the presence of functional CRH receptors in the myometrium suggests that CRH may modulate myometrial contractility and hence parturition. CRH action is mediated via multiple receptor subtypes and pregnancy results in differential receptor expression. These receptors are primarily linked to the adenylate cyclase second messenger system, which would help the intracellular microenvironment to maintain the required myometrial quiescence. CRH can exert further actions such as inhibition of prostaglandin production to prevent contractions. At term under the influence of oxytocin there is a modification in the coupling mechanisms that leads to a decrease in the biological activity of the CRH receptor and in the generation of cyclic adenosine monophosphate which favours myometrial contractions. CRH, via distinct receptor subtypes, is then able to enhance the contractile response of the myometrium. This hypothesis places CRH in a central role in coordinating the smooth transition from a state of relaxation to one of contraction.

Rat cerebral cortex corticotropin-releasing hormone receptors: evidence for receptor coupling to multiple G-proteins

The wide distribution of corticotrophin‐releasing hormone (CRH) receptors in brain and periphery appear to be important in integrating the responses of the brain, endocrine and immune systems to physiological, psychological and immunological stimuli. The type 1 receptors are highly expressed throughout the cerebral cortex, a region involved in cognitive function and modulation of stress responses, where they are coupled to the adenylyl cyclase system. Using techniques that analyse receptor‐mediated guanine‐nucleotide binding protein (G‐proteins) activation, we recently demonstrated that expressed type 1α CRH receptors are capable of activating multiple G‐proteins, which suggests that CRH can regulate multiple signalling pathways. In an effort to characterize the intracellular signals generated by CRH in the rat cerebral cortex we sought to identify G‐proteins activated by CRH in a physiological membrane environment. Rat cerebral cortical membrane suspensions were analysed for the ability of CRH to stimulate incorporation of [α‐32P]‐GTP‐γ‐azidoanilide to various G‐protein α‐chains. Our results show that CRH receptors are coupled to and activate at least five different G‐proteins (Gs, Gi, Gq/11, Go and Gz) with subsequent stimulation of at least two intracellular signalling cascades. In addition, the photoaffinity experiments indicated that the CRH receptors preferentially activate the 45u2003kDa form of the Gsα‐protein. This data may help elucidate the intracellular signalling pathways mediating the multiple actions of CRH especially under different physiological conditions.

SUPEROXIDE ELECTRODE BASED ON COVALENTLY IMMOBILIZED CYTOCHROME C : MODELLING STUDIES

We have recently described an optimised electrode for the detection of enzymatic and cellular superoxide (O2*-) production based on cytochrome c immobilized covalently at a surface-modified gold electrode and applied this system to the study of free radical production by activated human glioblastoma cells. In this paper we report the development of a mathematical model for the O2*- electrode responding to enzymically produced O2*- which should enable the determination of absolute concentrations of O2*- in biological systems when this electrode is employed for direct, real-time monitoring of free radical release and interactions.

Potential signalling pathways underlying corticotrophin‐releasing hormone‐mediated neuroprotection from excitotoxicity in rat hippocampus

In several neurological disorders including cerebral ischaemia, glutamate has been implicated as a neurotoxic agent in the mechanisms leading to neuronal cell death. The role of corticotrophin‐releasing hormone (CRH), the 41‐amino acid peptide, which activates the HPA axis in response to stressful stimuli, remains controversial. In this study, we report that CRH in low physiological concentrations (2u2003pm), prevented glutamate‐induced neurotoxicity via receptor‐mediated mechanisms when administered to organotypic hippocampal cultures both during and after the glutamate‐induced insult. Detailed investigations on the mechanisms mediating this neuroprotective effect showed that activation of the adenylate cyclase pathway and induction of MAP kinase phosphorylation mediate the CRH action. In addition we showed that CRH can inhibit the phosphorylation of JNK/SAPK by glutamate. Most importantly, we showed that CRH can afford neuroprotection against neurotoxicity up to 12 h following the insult, suggesting that CRH is acting at a late stage in the neuronal death cycle, and this might be important in the development of novel neuroprotective agents in order to improve neuronal survival following the insult.

DIRECT, REAL-TIME SENSING OF FREE RADICAL PRODUCTION BY ACTIVATED HUMAN GLIOBLASTOMA CELLS

Primary brain injury initiates a cascade of events which result in secondary brain damage. Although, at present, the biochemical and molecular mechanisms of nerve cell death are not well understood, sufficient evidence now exists to implicate free radicals in this brain injury response. In the light of the current understanding on the role of free radicals in cell mortality, we report on the use of two specific sensors, which we use to measure the direct, simultaneous and real time electrochemical detection of both superoxide (O2.-) and nitric oxide (NO), produced by activated glioblastoma cells. The development and application of these novel methods has enabled us to show that both the cytokine-mediated induction of the enzymes responsible for the generation of these radical species, and the metabolic requirements of the cell can modulate cell messenger release. Importantly, the data collected provides dynamic information on the time course of free radical production, as well as their interactions and their involvement in the process of cell death. In particular, one of the major advances afforded by this technology is the demonstration that suppression of one of either of the two cellular generated radical species (NO and O2.-) leads directly to a corresponding increase in the species that was not being deliberately inhibited or scavenged. This finding may indicate a mechanism involving inter-enzyme regulation of free radical production in glial cells (a phenomenon which may, in future, also be shown to operate in other relevant cell models).

Up-regulation of nitric oxide synthase and modulation of the guanylate cyclase activity by corticotropin-releasing hormone but not urocortin II or urocortin III in cultured human pregnant myometrial cells

The biological actions of corticotropin-releasing hormone (CRH) in the human myometrium during pregnancy and labor are unknown. We hypothesized that CRH may modulate the nitric oxide system, and influence myometrial relaxation/contractility. Incubation of myometrial cells with CRH, but not urocortin II or urocortin III, for 8–16 h significantly induced mRNA and protein expression of endothelial and brain but not inducible nitric oxide synthase (NOS) isoforms. This action resulted in increased activity of soluble guanylate cyclase (GCs), demonstrated by the enhanced cGMP-producing capacity of the NO donor, sodium nitroprusside. CRH also caused acute activation of the membrane-bound GC, shown by increased basal or atrial natriuretic peptide (ANP)-stimulated cGMP production. These effects appeared to be mediated via the R1 receptors because the CRH receptor antagonists, astressin and antalarmin but not anti-sauvagine 30, could block them. The acute effects of CRH were significantly reduced by inhibition of protein kinase A (PKA) activity, suggesting it is partially PKA dependant. Activation of protein kinase C (PKC) resulted in significant inhibition of both ANP-and CRH-stimulated cGMP production, suggesting a direct effect of PKC on membrane-bound GC. In conclusion, CRH appears to have a dual effect on myometrial NOS/GC pathway, a short term effect predominantly mediated by PKA, and a long-term effect increasing constitutive NOS expression, mediated by a PKA-independent mechanism. This mechanism could potentially be active during human pregnancy, and, because cGMP stimulates myometrial relaxation, these findings further suggest that during pregnancy CRH primarily activates intracellular signals that contribute to the maintenance of myometrial quiescence.

Novel Insights into the Cardio-Protective Effects of FGF21 in Lean and Obese Rat Hearts

Aims Fibroblast growth factor 21 (FGF21) is a hepatic metabolic regulator with pleotropic actions. Its plasma concentrations are increased in obesity and diabetes; states associated with an increased incidence of cardiovascular disease. We therefore investigated the direct effect of FGF21 on cardio-protection in obese and lean hearts in response to ischemia. Methods and Results FGF21, FGF21-receptor 1 (FGFR1) and beta-Klotho (βKlotho) were expressed in rodent, human hearts and primary rat cardiomyocytes. Cardiac FGF21 was expressed and secreted (real time RT-PCR/western blot and ELISA) in an autocrine-paracrine manner, in response to obesity and hypoxia, involving FGFR1-βKlotho components. Cardiac-FGF21 expression and secretion were increased in response to global ischemia. In contrast βKlotho was reduced in obese hearts. In isolated adult rat cardiomyocytes, FGF21 activated PI3K/Akt (phosphatidylinositol 3-kinase/Akt), ERK1/2(extracellular signal-regulated kinase) and AMPK (AMP-activated protein kinase) pathways. In Langendorff perfused rat [adult male wild-type wistar] hearts, FGF21 administration induced significant cardio-protection and restoration of function following global ischemia. Inhibition of PI3K/Akt, AMPK, ERK1/2 and ROR-α (retinoic-acid receptor alpha) pathway led to significant decrease of FGF21 induced cardio-protection and restoration of cardiac function in response to global ischemia. More importantly, this cardio-protective response induced by FGF21 was reduced in obesity, although the cardiac expression profiles and circulating FGF21 levels were increased. Conclusion In an ex vivo Langendorff system, we show that FGF21 induced cardiac protection and restoration of cardiac function involving autocrine-paracrine pathways, with reduced effect in obesity. Collectively, our findings provide novel insights into FGF21-induced cardiac effects in obesity and ischemia.

Circadian rhythmicity of salivary leptin in healthy subjects

In humans, circulating leptin correlates with body weight and fat mass. The main source of leptin is adipose tissue although placenta has been shown to produce leptin. More recently leptin and its receptor have been found in gastric mucosa and salivary gland. In the present study, we set to confirm the presence of leptin in human saliva and salivary gland, and if present, investigate whether salivary leptin exhibited circadian rhythmicity. Saliva and plasma were collected at 2 h intervals for 24 h in 12 healthy volunteers (6 females; 6 males). As previously described in human saliva and salivary gland, we have confirmed the presence of leptin at both mRNA and protein level. Leptin concentrations were significantly higher in plasma than saliva (p = 0.002), and a strong correlation between salivary and plasma leptin was demonstrated (r = 0.76; p < 0.001). Salivary leptin, like plasma leptin, showed circadian variations in both men and women, with a peak around 2400 h and a nadir at 1000 h. Both, mean 24-h salivary leptin and plasma leptin concentrations were significantly higher in women than men (p < 0.05; p < 0.01, respectively), but the ratios of salivary and plasma leptin concentrations were higher in men (p < 0.05), independent of other variables measured. In this study we have confirmed that leptin is synthesised and secreted by the human salivary gland, and have demonstrated for the first time that salivary leptin shows circadian variation. The precise role of salivary leptin needs to be elucidated. Our findings suggest that there may be differential regulation of leptin (salivary) between men and women. Finally, measurement of leptin in saliva is a simple, non-invasive and may be an acceptable alternative to plasma sampling.