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

AIMS/HYPOTHESIS: Pregnancy, 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. METHODS: Real-time RT-PCR, radioimmunoassay, Western blotting, radioligand binding and immunofluorescent analyses were applied to demonstrate the expression, secretion and functionality of placental adiponectin. RESULTS: Adiponectin 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-alpha, IFN-gamma, 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/INTERPRETATION: Our 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.

Evaluation of delivery of enhanced diabetes care to patients of South Asian ethnicity: the United Kingdom Asian Diabetes Study (UKADS).

AIMS: We tested the hypothesis that enhanced care for diabetes, tailored to the needs of the South Asian community with Type 2 diabetes, would improve risk factors for diabetic vascular complications and ultimately reduce morbidity and mortality. PATIENTS AND METHODS: The study was a cluster randomized controlled trial (RCT) with general practice the unit of randomization. Six West Midlands general practices with a high proportion of South Asian patients were randomized to 'enhanced care' using Asian link workers and extra community diabetes specialist nurse sessions (intervention) or continued standard practice care (control). RESULTS: Of 401 patients recruited to the study, 361 (90%), comprising 178 from Coventry and 183 from Birmingham were eligible and included in the analyses. The mean age at baseline (standard deviation, SD) was 58.9 (11.7 years) with median (interquartile range; IQR) duration of diabetes 6.5 (3-11) years. At one year follow-up there was a significant difference in reduction of systolic (4.6 mmHg, P = 0.035) and diastolic blood pressure (3.4 mmHg, P = 0.003) and total cholesterol (0.4 mmol/l, P = 0.005), comparing the intervention and control groups. After adjusting for baseline measurement and age, only differential reduction in diastolic blood pressure remained significant. There was no significant change in HbA(1c) and no difference between the groups. CONCLUSIONS: Using link workers and extra community diabetes specialist nurse input together with treatment protocols in primary care might prove a useful strategy in working towards NSF targets for diabetes management. In this study, small reductions in blood pressure and cholesterol were achieved. Improvement in glycaemic control may require longer and possibly different strategies. Further research is required to evaluate fully the effectiveness, including the costs and longer term sustainability of culturally sensitive initiatives.

Expression and signalling characteristics of the corticotrophin-releasing hormone receptors during the implantation phase in the human endometrium.

Corticotrophin-releasing hormone (CRH) has been identified in several peripheral tissues, including the female reproductive organs. CRH is expressed in the placenta, myometrium, epithelial endometrium and the endometrial stromal cells at all phases of the menstrual cycle. Similarly, CRH receptors are present in pregnant and non-pregnant myometrium, placenta and endometrium. Putative roles of CRH in the endometrium include involvement in implantation, decidualisation and maintenance of pregnancy. In this study we sought to investigate in detail the CRH receptor repertoire expressed in the human endometrium and their signalling characteristics. Using RT-PCR we were able to demonstrate the expression of CRH receptor 1alpha (CRH-R1alpha) and CRH-R2alpha in the human endometrium. CRH-R1beta was present in 40% of endometrial cDNAs examined. No apparent expression of CRH-R2beta, CRH-R2gamma or any other CRH-R1 splice variants was detected. Chemical cross-linking studies with 125I-ovine CRH revealed that the endometrial CRH receptor has a molecular weight of 45 kDa. Using the non-hydrolysable photoreactive analogue [alpha-32P]GTP-azidoanilide and peptide antisera raised against G-protein alpha-subunits, we then studied coupling of endometrial CRH receptors to G proteins. Treatment of endometrial membranes with human CRH (100 nM) increased the labelling of Gq and Gs, but not Gi or Go. These results were supported by experiments in epithelial cells of the non-pregnant human endometrium in the secretory phase which showed that CRH induced increases in both cAMP and inositol trisphosphate levels. These results suggested that CRH may exert multiple effects in the human endometrium via distinct signalling cascades. These events are possibly mediated via different receptor subtypes.

Measurement of thyroglobulin mRNA in peripheral blood as an adjunctive test for monitoring thyroid cancer.

AIMS: Monitoring treated patients with thyroid cancer for recurrent or metastatic disease is currently based upon the serial measurement of circulating plasma thyroglobulin (Tg) concentrations. However, the clinical usefulness of Tg immunoassays is limited by poor sensitivity and interference from anti-Tg antibodies. This study investigated whether the detection of Tg mRNA in peripheral blood, using reverse transcriptase polymerase chain reaction (RT-PCR), is of value in the biochemical surveillance of patients with thyroid cancer. METHODS: RNA was extracted from peripheral blood of five normal controls, six patients with abnormal thyroid function tests, and 28 patients who had undergone thyroidectomy for well differentiated thyroid cancer. From each, an 87 bp product from base pair 262 to 348 in the cDNA sequence of the thyroglobulin gene was amplified by RT-PCR. RESULTS: Tg mRNA was detected in normal individuals and patients with thyroid cancer. In the group of patients studied, identification of metastatic thyroid tissue by radioiodine scanning correlated better with Tg mRNA assay results than with serum Tg concentrations (accuracy 84% v 75%). No interference from circulating Tg antibodies was apparent. In patients studied prospectively over a 12 month period, there was a significant correlation between detectable Tg mRNA in peripheral blood and the presence or absence of metastatic disease, as demonstrated by radioiodine scanning. CONCLUSIONS: These results suggest that detection of Tg mRNA in blood is a more sensitive marker for metastatic thyroid disease than Tg immunoassay, and appears to be unaffected by the presence of circulating anti-Tg antibodies.

Reduced expression of corticotropin-releasing hormone receptor type-1 alpha in human preeclamptic and growth-restricted placentas.

Placentally derived CRH seems to play a major role in the mechanisms controlling human pregnancy and parturition, via activation of specific receptors widespread in reproductive tissues. In the human placenta, CRH seems to modulate vasodilation, prostaglandin production, and ACTH secretion. It has also been suggested that CRH might act as a placental clock, determining the length of gestation. In addition, maternal plasma CRH concentrations are further elevated in pregnancies associated with abnormal placental function, such as preeclampsia and intrauterine growth retardation (IUGR). In this study, we sought to investigate the expression of CRH-R1 alpha levels in placentas from women who have undergone normal deliveries (control group) and patients who have been diagnosed as having preeclampsia or IUGR. Results showed that placental CRH-R1 alpha mRNA levels (as shown by quantitative RT-PCR) and protein levels (shown by Western blotting analysis) were significantly (P < 0.05) reduced in all of the complicated pregnancies. In contrast, levels of the angiotensin II receptor were elevated in preeclampsia and reduced in IUGR subjects, as shown by RT-PCR and Western blotting analysis. These findings might suggest that changes in receptor expression may contribute toward dysregulation of the dynamic balance controlling vascular resistance.

Corticotropin-releasing hormone receptors.

Corticotropin-releasing hormone (CRH) and related peptides (urocortins, sauvagine, urotensin) play a central role in the co-ordination of autonomic, behavioural, cardiovascular, immune and endocrine responses to stressful stimuli. Their actions are mediated through activation of two types of G-protein-coupled receptors encoded by separate genes. In this review we focus on the diverse structural and functional characteristics of the family of CRH-like peptides and their receptors.

Steroids mediate the expression of cytoplasmic and membrane-linked components in human myometrial cells.

It is well known that the smooth muscle of the human myometrium is a target for the steroid hormones progesterone (P4) and estrogen. Progesterone is believed to participate in the maintenance of pregnancy, while estrogen is possibly involved in the process of parturition by promoting cervical dilatation. We examined the combined effects of P4 and 17beta-estradiol (E2) on components of signalling pathways in human myometrial cells in vitro by immunoblotting. Long-term treatment of myometrial cells with a series of concentrations of P4 and E2 in combination caused a change in the phosphorylation status of p42/44 mitogen-activated protein kinase and of c-Jun N-terminal kinase (SAPK/JNK). P4 and E2 caused a decrease in protein expression of Gqalpha, Gzalpha, Gi1/2alpha and, to a lesser extent, G0alpha. The two steroids caused a decrease in the expression of the two small GSalpha isoforms. Cyclo-oxygenase-2 expression was increased by 2.5-fold after steroid treatment, while proliferating cell nuclear antigen expression levels remained unchanged. These observations show that the combination of P4 and E2 influences intracellular and membrane-bound components of signal transduction pathways in human myometrial cells. The implications of the two steroid hormones on intracellular signalling pathways in the human myometrium merits further investigation.

Expression of orexin-A and functional orexin type 2 receptors in the human adult adrenals: implications for adrenal function and energy homeostasis.

The hypothalamic peptides, orexin-A and orexin-B, have been implicated in the regulation of feeding behavior. In starved rats catabolic activity quickly predominates, reinforced by elevated corticosterone, independent of ACTH, implicating adrenal activity as a metabolic regulator. In view of these findings, we investigated whether orexin and orexin receptors are present in human adult adrenals and might therefore be implicated in hormonal regulation and energy homeostasis outside the central nervous system. RT-PCR, fluorescent in situ hybridization, immunoblotting, and immunostaining analysis confirmed the expression of the orexin type 2 receptor, but not of orexin type 1 receptor, in the adrenal cortex. Immunoblotting analysis also detected the presence of the prepro-orexin and its cleaved product orexin-A. Treatment of adult adrenal membranes with orexin-A increased the labeling of G(s), G(q), and, to a lesser degree, G(i), but not G(o). Stimulation with orexin-A induced cAMP and IP3 production in a dose-dependent manner. The data presented here provide conclusive evidence for the presence of orexin-A and orexin type 2 receptors in human adult adrenal glands. At the moment the functional relevance of this is uncertain. However, it is known that both orexin-A and orexin-B can induce corticosterone production in dispersed rat adrenocortical cells. Our data provide further evidence for a functional link between orexogenic signals and adrenal function. The concept that the peptide acting via these receptors in the adult adrenal is responsible for steroidogenesis and energy balance is attractive.

Expression and coupling characteristics of the CRH and orexin type 2 receptors in human fetal adrenals.

Hormones produced by the fetal adrenal regulate fetal growth, steroidogenic activity, and intrauterine homeostasis, which are essential for the maintenance of pregnancy and the preparation of the fetus for extrauterine life. There is a functional interaction between CRH and the fetal adrenal, as CRH increases dehydroepiandrosterone sulfate production in cultured fetal adrenal cells. Moreover, in a rodent model administration of orexin A induced corticosterone production. To examine this relationship in more detail we measured the expression of the different subtypes of CRH and orexin receptors and their specific coupling to G protein alpha-subunits upon activation with CRH and orexin A, respectively. Using RT-PCR and fluorescent in situ hybridization analysis, we demonstrated the presence of CRH receptors 1alpha and 2alpha, and orexin type 2 receptor mRNA. None of the other CRH receptor variants or orexin type 1 receptor were detected. Immunofluorescent analysis and Western blotting confirmed the protein expression of both receptors, which also bind fluo-CRH and fluo-orexin with high affinity. Immunoblotting analysis confirmed the expression of prepro-orexin and orexin A in fetal adrenals. Using photoaffinity labeling, we determined which G proteins are coupled to the CRH and orexin receptors in fetal adrenals when challenged with CRH or orexin. Treatment of fetal adrenal membranes with CRH (100 nM) increased the labeling of G(o) and, to a lesser extent, G(s), but not G(i) and G(q), whereas treatment with orexin A (100 nM) increased the labeling of G(s) and G(i), but not G(o) and G(q). These findings provide new insights into the components of the signal transduction machinery in human fetal adrenals and demonstrate for the first time the presence of functional orexin receptors outside of the CNS in humans.

Developmental expression and characterization of FS39, a testis complementary DNA encoding an intermediate filament-related protein of the sperm fibrous sheath.

Proteins immunologically related to intermediate filaments have been identified in the sperm fibrous sheath but remain uncharacterized. We isolated and characterized a novel intermediate filament-related protein (FS39) localized to the fibrous sheath of the sperm tail. We used Northern blot analysis to establish that FS39 is transcribed predominantly in the testis of mice >18-20 days old. At this age, spermatogenesis has proceeded to the development of the first round haploid spermatids. In situ hybridization revealed that FS39 mRNA is first detectable in late step 3 spermatids, is at its highest level during steps 9 and 10, and diminishes in steps 13 and 14. Western blot analysis identified a single protein of 39 kDa in mouse and rat testis and epididymis, suggesting the protein is conserved in rodents. Indirect immunofluorescence localized FS39 to the fibrous sheath of the sperm tail, and in testis sections expression was detected from step 13 and step 14 spermatids onward, indicating FS39 is under translational control. Southern blot analysis showed FS39 to be a single copy gene, and hybridization to human genomic DNA suggested that a human equivalent gene is present. These results demonstrate that FS39 is transcribed in testis tissue during the haploid phase of spermatogenesis, is present in mature sperm, and codes for a novel 39-kDa intermediate filament-related protein of the fibrous sheath.

The role of corticotropin-releasing hormone receptors in placenta and fetal membranes during human pregnancy.

Corticotropin-releasing hormone (CRH) is a 41 amino acid polypeptide that exerts a wide spectrum of hypothalamic and extrahypothalamic functions. Moreover, the placenta and other intrauterine tissues produce and secrete immunoreactive CRH. It has been demonstrated that placental CRH is secreted into the maternal circulation in large amounts during the third trimester of human pregnancy and may play an important role in the onset of labor. CRH exerts a number of functions within the intratuterine environment like induction of prostaglandin production and maintenance of the placental blood flow. Here we present an overview of current knowledge about the CRH receptor subtypes and their signaling properties within the feto-placental unit.

Effects of insulin and glucocorticoids on the leptin system are mediated through free leptin.

OBJECTIVE: Insulin and glucocorticoids are known to increase total leptin levels. However, the effects of insulin and glucocorticoids on the components of the leptin system - free leptin (FL), bound leptin (BL) and soluble leptin receptor (SR) - have not been elucidated. The aim of this study was to determine if there is a differential effect of insulin and glucocorticoids on the leptin system. MATERIAL AND METHODS: In the first of two studies (study 1), we measured free leptin (FL), bound leptin (BL), a soluble leptin receptor (SR) and insulin, by specific RIA methods, in six healthy subjects on a control day, and subsequently during a hyperinsulinaemic euglycaemic clamp study. In the second study (study 2) we measured the same parameters in six healthy subjects, before (day 1) and during administration of dexamethasone over 3 consecutive days. RESULTS: In study 1, on the control day FL levels rose over the 16 h monitoring period (P = 0.057) and SR levels declined (P < 0.001), but there was no change of BL levels. Even after accounting for diurnal variation, FL levels increased even more substantially over 12 h of insulin infusion than observed on the control day (P < 0.001). In study 2, mean FL concentration doubled from day 1 to day 2 (P = 0.01) and remained elevated subsequently. In contrast to FL, BL and SR levels remained unchanged during the study. Fasting insulin levels (pmol/l) increased from day 1 to day 2, but this rise only approached significance on day 4 (P = 0.05). CONCLUSION: We conclude that insulin and dexamethasone increase free leptin levels, but do not change the concentrations of bound leptin and soluble leptin receptor. Furthermore, the dexamethasone-induced rise in leptin levels is (at least partially) independent of the effects of glucocorticoid-induced hyperinsulinaemia.

Fatal emphysematous pyelonephritis with gas in the spinal extradural space in a patient with diabetes.

BACKGROUND: Emphysematous pyelonephritis (EP) is a rare but life-threatening condition of the upper urinary tract, characterized by the presence of gas in the renal parenchyma and perirenal space. The vast majority of patients with EP (90%) are known to have diabetes, with Escherichia coli being the most common causative pathogen. CASE REPORT: We present a case of fatal bilateral EP in a patient with diabetes, with an unusual radiological finding of gas around the spinal cord and in the psoas muscle, with renal parenchymal sparing. Our case serves as an important reminder of this life threatening entity in diabetic patients, which is not well recognized by clinicians because of its rarity.

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 1alpha 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 [alpha-32P]-GTP-gamma-azidoanilide to various G-protein alpha-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 45 kDa form of the Gs alpha-protein. This data may help elucidate the intracellular signalling pathways mediating the multiple actions of CRH especially under different physiological conditions.

Characterising the corticotropin-releasing hormone (CRH) receptors mediating CRH and urocortin actions during human pregnancy and labour.

The mechanism of human labour remains unresolved. One of the most important regulatory signals, however, appears to be corticotropin-releasing hormone (CRH), a hypothalamic peptide that controls the body's response to stress, which is also produced by the placenta and intrauterine tissues during pregnancy. CRH belongs to a family of peptides that includes urocortin, which shares sequence homology with CRH and is also expressed by the placenta and intrauterine tissues. During human pregnancy circulating CRH appears to have five main target tissues: the myometrium, the placenta, the fetal membranes, the fetal adrenal cortex and the vasculature. In these tissues CRH plays a role in the control of myometrial contractility,placenta vasodilation, peptide and prostaglandin production and adrenal steroidogenesis and probably many more, yet unidentified processes. The actions of CRH in these tissues are mediated via specific G-protein coupled membrane-bound receptors. These receptors have different functional characteristics, depending on where they are expressed and on the stage of pregnancy. In addition, their function depends upon other intracellular signals via communication between signalling cascades. These findings led us to propose a hypothesis for a dual role of CRH and other CRH-like peptides during pregnancy and labour.

Urocortin, but not corticotropin-releasing hormone (CRH), activates the mitogen-activated protein kinase signal transduction pathway in human pregnant myometrium: an effect mediated via R1alpha and R2beta CRH receptor subtypes and stimulation of Gq-proteins.

CRH and CRH-related peptides such as urocortin mediate their actions in the human myometrium via activation of two distinct classes of CRH receptors, R1 and R2. These heptahelical receptors are able to stimulate a number of different intracellular signals; one key mediator of G protein-activated intracellular signaling is the cascade of p42/p44, mitogen-activated protein kinase (MAPK). We therefore hypothesized that activation of MAPK might mediate CRH and or/urocortin actions in the myometrium. In cultured human pregnant myometrial cells, urocortin but not CRH was able to induce MAPK phosphorylation and activation, suggesting that in the human myometrium these two peptides have distinct actions and biological roles. To identify the particular receptor subtypes mediating this phenomenon, all known CRH receptors present in the human myometrial cells were stably expressed individually in HEK293 and CHO cells, and their ability to activate MAPK was tested. The R1alpha and R2beta, but not the R1beta, R1c, or R1d, receptor subtypes were able to mediate urocortin-induced MAPK activation. The signaling components were further investigated; activation of Gs, Go, or Gi proteins did not appear to be involved, but activation of Gq with subsequent production of inositol triphosphates (IP3) and protein kinase C (PKC) activation correlated with MAPK phosphorylation. Studies on Gq protein activation using [alpha-32P]-GTP-gamma-azidoanilide and IP3 production in cells expressing the R1alpha or R2beta CRH receptors demonstrated that urocortin was 10 times more potent than CRH. Moreover, urocortin (UCN) generated peak responses that were 50-70% greater than CRH in activating the Gq protein and stimulating IP3 production. In conclusion, UCN acting thought multiple receptor subtypes can stimulate myometrial MAPK via induction of the Gq/phospholipase C/IP3/PKC pathway, whereas CRH-induced activation of this pathway appears to be insufficient to achieve MAPK activation.

Fatal cholestatic jaundice associated with amitriptyline.

A 75-year-old woman was admitted with a two-week history of anorexia and vague abdominal pain. She had been taking amitriptyline 75 mg at night for depression for four months before her admission. On presentation she was jaundiced, but with no stigmata of chronic liver disease. Initial liver function tests showed a slightly raised bilirubin, but were otherwise normal. Over the next three weeks her bilirubin concentration continued to rise without evidence of biliary obstruction on ultrasound examination. Her condition continued to deteriorate, and she later developed renal failure consistent with hepatorenal syndrome. Seven weeks after admission she died following a large gastrointestinal bleed. At autopsy, liver histology confirmed pure cholestasis consistent with amitriptyline ingestion.