Calcitonin Gene Related Peptide, Adrenomedullin, and Adrenomedullin 2 Function in Uterine Artery During Human Pregnancy.

RATIONALE: Calcitonin gene-related peptide (CGRP) and its family members adrenomedullin (ADM) and adrenomedullin 2 (ADM2; also known as intermedin) support vascular adaptions in rat pregnancy. OBJECTIVE: This study aimed to assess the relaxation response of uterine artery (UA) for CGRP, ADM, and ADM2 in nonpregnant and pregnant women and identify the involved mechanisms. FINDINGS: (1) Segments of UA from nonpregnant women that were precontracted with U46619 (1µM) in vitro are insensitive to the hypotensive effects of CGRP, ADM, and ADM2; (2) CGRP, ADM, and ADM2 (0.1-100nM) dose dependently relax UA segments from pregnant women with efficacy for CGRP > ADM = ADM2; (3) the relaxation responses to CGRP, ADM, and ADM2 are differentially affected by the inhibitors of nitric oxide (NO) synthase (L-NAME), adenylyl cyclase (SQ22536), apamin, and charybdotoxin; (4) UA smooth muscle cells (UASMC) express mRNA for calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein (RAMP)1 and RAMP2 but not RAMP3; (5) receptor heterodimer comprising CRLR/RAMP1 and CRLR/RAMP2 but not CRLR/RAMP3 is present in UA; (6) soluble fms-like tyrosine kinase (sFLT-1) and TNF-α treatment decrease the expression of RAMP1 mRNA (P < 0.05) in UASMC; and (7) sFLT-1 treatment impairs the association of CRLR with all 3 peptides while TNF-α inhibits the interaction of CGRP but not ADM or ADM2 with CRLR in UASMC (P < 0.05). CONCLUSIONS: Relaxation sensitivity of UA for CGRP, ADM, and ADM2 is increased during pregnancy via peptide-specific involvement of NO system and endothelium-derived hyperpolarizing factors; vascular disruptors such as sFLT-1 and TNFα adversely impact their receptor system in UASMC.

Soluble fms-like tyrosine kinase-1 and angiotensin2 target calcitonin gene-related peptide family peptides in maternal vascular smooth muscle cells in pregnancy†.

Calcitonin gene-related peptide (CALCB), adrenomedullin (ADM), and adrenomedullin2 (ADM2) are hypotensive peptides that belong to CALCB family of peptides. Goal of this study was to identify the effect of fms-like tyrosine kinase (sFLT-1) and angiotensin2 (Ang2) on the function of these peptides in OA smooth muscle cells (OASMC) and assess the sensitivity of OA for these peptides in preeclampsia (PE) and normotensive pregnancy. METHODS: Peptide function was assessed by Cyclic adenosine monophosphate (cAMP) assays and wire myograph; mRNA expression by Polymerase chain reaction (PCR) and protein-protein interaction by proximity ligation assay and co-immunoprecipitation. FINDINGS: All three peptides increased cAMP synthesis in the order of efficacy CALCB > ADM = ADM2 and vascular endothelial growth factor (VEGF) mRNA in OASMC (P < 0.05); sFLT-1 mediated decrease in cAMP synthesis (P < 0.05) is differentially rescued by all three CALCB family peptides in OASMC (P < 0.005); sFLT-1 decreased receptor activity-modifying protein (RAMP)1 and RAMP2 mRNA expression (P < 0.05); Ang2 decreased the expression of calcitonin-receptor-like receptor and RAMP1 mRNA and desensitized CALCB and ADM2 receptors in OASMC (P < 0.05); sFLT-1 increased RAMP1and Ang2 type 1 receptor (AT1R) interaction in OASMC which is inhibited in presence of all three peptides; and all three peptides relax OA in PE with enhanced ADM2 response (P < 0.05). CONCLUSION: sFLT-1 and Ang2 impair OASMC mediated functional responses of CALCB family peptides which can be inhibited by respective peptide treatment. The sensitivity of OA for CALCB, ADM, and ADM2-mediated relaxation is retained in PE.

In utero low-protein-diet-programmed type 2 diabetes in adult offspring is mediated by sex hormones in rats†.

Sex steroids regulate insulin sensitivity and glucose metabolism. We had characterized a lean type 2 diabetes (T2D) rat model using gestational low-protein (LP) diet programming. Our objective was to identify if endocrine dysfunction leading to decreased sex hormone levels will precede the development of T2D and if steroid replacement will prevent the onset of the disease. Pregnant rats were fed control or isocaloric LP diet from gestational day 4 until delivery. Normal diet was given to all mothers after delivery and to pups after weaning. LP offspring developed glucose intolerance and insulin resistance at 4 months. We measured sex steroid hormone profiles and expression of key genes involved in steroidogenesis in testis and ovary. Furthermore, one-month old rats were implanted with 90-day slow release T and E2 pellets for males and females, respectively. Glucose tolerance test (GTT) and euglycemic hyperinsulinemic clamp was performed at 4 months. LP-programmed T2D males had low T levels and females had low E2 levels due to dysregulated gene expression during steroidogenesis in gonads. GTT and euglycemic hyperinsulinemic clamp showed that LP males and females were glucose intolerant and insulin resistant; however, steroid supplementation prevented the onset of glucose intolerance and insulin resistance. Rats that developed T2D by LP programming have compromised gonadal steroidogenesis leading to low T and E2 in males and females, respectively. Sex steroid supplementation prevented the onset of glucose intolerance and insulin resistance indicating low sex steroid levels could cause compromised glucose metabolism ultimately leading to T2D.

Complement inhibitor Crry expression in mouse placenta is essential for maintaining normal blood pressure and fetal growth.

Many circumstantial evidences from human and animal studies suggest that complement cascade dysregulation may play an important role in pregnancy associated complications including preeclampsia. Deletion of rodent specific complement inhibitor gene, Complement Receptor 1-related Gene/Protein y (Crry) produces embryonic lethal phenotype due to complement activation. It is not clear if decreased expression of Crry during pregnancy produces hypertensive phenotype. We downregulated Crry in placenta by injecting inducible lentivialshRNA vectors into uterine horn of pregnant C57BL/6 mice at the time of blastocyst hatching. Placenta specific downregulation of Crry without significant loss of embryos was achieved upon induction of shRNA using an optimal doxycycline dose at mid gestation. Crry downregulation resulted in placental complement deposition. Late-gestation measurements showed that fetal weights were reduced and blood pressure increased in pregnant mice upon downregulation of Crry suggesting a critical role for Crry in fetal growth and blood pressure regulation.

Common variants of fetal and maternal complement genes in preeclampsia: pregnancy specific complotype.

Preeclampsia (PE) is a pregnancy specific hypertensive disorder. If untreated PE leads to life threatening condition, eclampsia. Systemic complement activation levels are increased during pregnancy compared to non-pregnant women of childbearing age. In PE, systemic complement levels are further increased, and higher complement deposition has been observed on placentas. We hypothesize that combinations of common SNPs in maternal and fetal complement genes constitute pregnancy specific complotypes and predispose women to PE. In this study, we sequenced two maternal (factor H and C3) and one fetal (CD46) complement genes and identified a total of 9 common SNPs. Minor allele frequencies of two fetal CD46 SNPs were significantly higher in PE. Further, complotypes consisting of fetal CD46 variants and maternal CFH/C3 variants were highly prevalent in PE patients compared to normotensive pregnancies. Placental complement deposition and maternal alternative pathway 50 (AP50) values were higher in PE pregnancies. Irrespective of disease status, two CD46 variants were associated with reduced placental CD46 expression and one CFH variant was associated with increased maternal AP50 values.

Sex Dependent Dysregulation of Hepatic Glucose Production in Lean Type 2 Diabetic Rats.

We have characterized a lean type 2 diabetic rat model by gestational low protein programming. We aimed to identify if the regulation of hepatic glucose production (HGP) via gluconeogenesis and glycogenolysis is affected and if there are any sex differences. Fasting (6-7 months old) type 2 diabetic rats received 2H2O followed by a primed constant rate infusion of [6,6-2H2] glucose. Blood samples were drawn during steady states after 4 h of fasting and following a euglycemic hyperinsulinemic clamp. HGP and the fraction of glucose derived from gluconeogenesis under fasting and euglycemic states were measured from steady state glucose enrichments after the infusion of [6,6-2H2]glucose and 2H2O tracers. Glycogenolysis was determined by calculating the difference between total HGP and gluconeogenesis rates. Hepatic gene expression of enzymes involved in HGP were quantified using qPCR. HGP rates was similar during fasting in both groups and sexes. However, under simulated fed condition, HGP rate was suppressed in controls but not in type 2 diabetic rats. They also showed inefficient HGP suppression in a simulated fed state. Differential analysis showed that suppression of both gluconeogenesis and glycogenolysis under simulated fed state was affected in these low protein programmed type 2 diabetic rats. These effects were greater in females when compared to males. Further, key genes involved in these processes like G6Pase, Pepck, pyruvate carboxylase, and glycogen phosphorylase in liver were dysregulated. Our data shows impaired suppression of HGP via gluconeogenesis and glycogenolysis in type 2 diabetic rats with greater effects on females.

Circulating Adrenomedullin Is Elevated in Gestational Diabetes and Its Role in Impaired Insulin Production by ß-Cells.

Context: Defective pancreatic ß-cell adaptation in pregnancy plays an important role in the pathophysiology of gestational diabetes mellitus (GDM), but the molecular basis remains unclear. Objectives of this study were to determine if circulating levels of adrenomedullin (ADM) in women with GDM are elevated and to assess the effects of ADM on insulin synthesis and secretion by human pancreatic ß-cells. Design: A stable gene product of ADM precursor, midregional pro-adrenomedullin (MR-proADM), was measured in plasma of pregnant women with normal glucose tolerance (NGT, n = 10) or GDM (n = 11). The ß-Lox5 cell line, derived from human pancreatic ß-cells, was transduced with homeodomain transcription factor pancreatic-duodenal homeobox (PDX) factor 1 (PDX1) encoding lentiviral vector and treated with different doses of ADM. mRNA for insulin, ADM, and its receptor components in ß-Lox5 cells and insulin in media were measured. Results: Plasma MR-proADM levels were significantly higher in GDM compared with patients with NGT. Pancreatic ß-Lox5 cells express mRNA for insulin, ADM, and its receptor components. PDX1 transduction and cell-cell contact synergistically promote ß-Lox5 cells insulin mRNA and secretion. Furthermore, ADM dose-dependently inhibited mRNA and secretion of insulin in ß-Lox5 cell aggregates. These inhibitory effects were blocked by ADM antagonist ADM22-52, cAMP-dependent protein kinase A inhibitor KT5720, and Erk inhibitor PD98059, but not by PI-3K the inhibitor wortmannin. Conclusions: Circulating ADM concentrations were elevated in pregnant women with GDM. ADM suppresses insulin synthesis and secretion by pancreatic ß-cells in vitro. Thus, increased circulating ADM may contribute to the defective adaptation of ß-cells in diabetic pregnancies, and blockade of ADM actions with its antagonists may improve ß-cell functions.

Upregulation and release of soluble fms-like tyrosine kinase receptor 1 mediated by complement activation in human syncytiotrophoblast cells.

PROBLEM: Antiangiogenic molecule soluble fms-like tyrosine kinase receptor 1 (sFLT1) released from trophoblast cells is associated with pregnancy-specific hypertensive disorder pre-eclampsia. Cause of elevated sFLT1 in pre-eclampsia patients is not well understood. Despite evidence of excess systemic and placental complement activation in pre-eclampsia patients, its role in pathophysiology is not clear. If the complement activation plays a role in upregulation and secretion of sFLT1 is not known. METHOD OF STUDY: Human trophoblast cells were isolated from term placentas and allowed to syncytialize. Complement was activated in vitro at sublethal levels on syncytiotrophoblast cells. Effect of complement activation on expression and release of sFLT1 was assessed by comparing its levels in these cells with and without complement activation. RESULTS: Sublethal level of complement activation on syncytialized human trophoblast cells induced upregulation of sFLT1 mRNA and protein. Complement also induced secretion of sFLT1 in a manner depending on degree of activation. Anaphylatoxins C3a induced upregulation but not the release of sFLT1. Release of terminal membrane attack complex (MAC) was associated with sFLT1 secretion. CONCLUSION: Complement activation plays a major role in both the expression and secretion of sFLT1 from syncytial trophoblast cells. The terminal MAC complex is involved in its secretion. Increased levels of sFLT1 in pre-eclampsia patients may be due to complement-induced upregulation and secretion.

Unexplored deep sea habitats in active volcanic Barren Island, Andaman and Nicobar Islands are sources of novel halophilic eubacteria.

Deep Sea sediment cores were collected from the surrounding of active volcanic Barren Island, Andaman & Nicobar Islands. A total of 24 halophilic eubacteria were isolated and identified based on their biochemical and 16S rDNA sequences. Three major classes (Gamma-Proteobacteria, Alpha-Proteobacteria and Bacilli) of bacteria were detected in the deep sea sediments of active volcanic Barren Island. Among those, 37% of isolates exhibited antimicrobial activity against all tested Gram positive and Gram negative clinical pathogens. 60% of isolates revealed the presence of either PKS or NRPS genes and 65% isolates disclosed medium to higher level of cytotoxicity in MB-231 breast cancer cell line. Majority of the isolates revealed excellent potential for bioprospecting of novel byproducts with industrial and pharmaceutical importance.

Folate treatment partially reverses gestational low-protein diet-induced glucose intolerance and the magnitude of reversal is age and sex dependent.

OBJECTIVES: Gestational low-protein (LP) programming causes glucose intolerance (GI) and insulin resistance (IR) in adult offspring. Folate supplementation has been shown to rescue the offspring from various programming effects. The aim of this study was to investigate whether folate supplementation during pregnancy reverses LP-induced GI and IR. METHODS: Pregnant rats were fed control (20% protein), isocaloric low-protein (LP, 6%) or LP with 5 mg/kg folate (LPF) diets from gestational day 4 to delivery. The control diet was given during lactation and to pups after weaning. Glucose tolerance test was done at 1, 2, and 3 mo of age followed by euglycemic-hyperinsulinemic clamp at 4 mo. Rats were sacrificed at 4 mo and their gonadal, renal, inguinal, brown fat, and pancreas were weighed and expressed relative to their body weight. RESULTS: LP- and LPF-fed dams showed similar weight loss during late pregnancy after decreased feed intake. Both LP and LPF pups were smaller at birth but their weights caught up like that of controls by 3 mo. In males, folate supplementation reduced LP-induced GI at 2 mo (glucose area under the curve [AUC]: 1940 mmol/L × 180 min in LP, 1629 mmol/L × 180 min in LPF, and 1653 mmol/L × 180 min in controls; P <0.05, LP versus control and P <0.01, LP versus LPF) but the effect diminished at 3 mo. In females, folate reduced GI at 1 mo (glucose AUC: 1406 mmol/L × 180 min in LP, 1264 mmol/L × 180 min in LPF, and 1281 mmol/L × 180 min in controls; P <0.05, LP versus control and LP versus LPF) but had no effect at 2 and 3 mo. Interestingly, the LPF group had higher pancreatic weights than other groups, suggesting that folate helps in pancreatic development enabling the LPF rats to produce/secrete more insulin to maintain euglycemia. Euglycemic-hyperinsulinemic clamp shows both LP and LPF are insulin resistant compared with controls by 4 mo with LPF more severe than LP in males. Interestingly, females were more insulin resistant than males. CONCLUSIONS: Folate treatment partially reverses LP-induced GI and the magnitude of reversal is age and sex dependent. Furthermore, folate treatment does not reverse IR in either sex but makes it worse in males at 4 mo. The present study demonstrated that folate treatment is not sufficient to rescue the LP programming effects.

Decreased insulin secretion in pregnant rats fed a low protein diet.

Low protein (LP) diet during pregnancy leads to reduced plasma insulin levels in rodents, but the underlying mechanisms remain unclear. Glucose is the primary insulin secretagogue, and enhanced glucose-stimulated insulin secretion (GSIS) in beta cells contributes to compensation for insulin resistance and maintenance of glucose homeostasis during pregnancy. In this study, we hypothesized that plasma insulin levels in pregnant rats fed LP diet are reduced due to disrupted GSIS of pancreatic islets. We first confirmed reduced plasma insulin levels, then investigated in vivo insulin secretion by glucose tolerance test and ex vivo GSIS of pancreatic islets in the presence of glucose at different doses, and KCl, glibenclamide, and L-arginine. Main findings include (1) plasma insulin levels were unaltered on day 10, but significantly reduced on days 14-22 of pregnancy in rats fed LP diet compared to those of control (CT) rats; (2) insulin sensitivity was unchanged, but glucose intolerance was more severe in pregnant rats fed LP diet; (3) GSIS in pancreatic islets was lower in LP rats compared to CT rats in the presence of glucose, KCl, and glibenclamide, and the response to L-arginine was abolished in LP rats; and (4) the total insulin content in pancreatic islets and expression of Ins2 were reduced in LP rats, but expression of Gcg was unaltered. These studies demonstrate that decreased GSIS in beta cells of LP rats contributes to reduced plasma insulin levels, which may lead to placental and fetal growth restriction and programs hypertension and other metabolic diseases in offspring.

Adrenomedullin2 (ADM2)/Intermedin (IMD): A Potential Role in the Pathophysiology of Preeclampsia.

CONTEXT: It is not known whether decreases in trophoblast invasion promoting the peptide, adrenomedullin2 (ADM2) system is associated with preeclampsia (PreE). OBJECTIVE: The objective of the study was to assess the changes in ADM2 levels in plasma, placenta, and amniotic fluid (AF) and its receptor components in placenta from PreE pregnancy compared with the age-matched normal and study the effect of ADM2 on the synthesis of nitric oxide (NO), endothelial nitric oxide synthase (eNOS), and matrix-metalloproteinase (MMP)-2 and MMP-9 in trophoblast cells. RESULTS: PreE is associated with a decreased expression of ADM2 in plasma and placenta (P < .05); ADM2 interacts with a seven-transmembrane G protein-coupled receptor, calcitonin receptor-like receptor (CRLR) in HTR-8/SVneo cells; placental expression of ADM2/CRLR complex is lower in PreE; mRNA for CRLR and receptor activity-modifying protein-3 are lower, whereas receptor activity-modifying protein-2 is higher in the PreE placenta (P < .05); ADM2 levels in the second trimester are lower in the AF from pregnant women who develop PreE later in gestation (P < .05); ADM2 is localized to the epithelium of the amnion and the ectoderm and mesoderm of the chorion in term fetal membranes; ADM2 increases NO production, eNOS, and MMP2/9-immunoreactivity, whereas ADM2 knockdown inhibits the expression of eNOS and MMP2/9 mRNA and S-nitrosylation in HTR-8/SVneo cells; and ADM2-induced increases in MMP2/9 activity is inhibited by L-nitro-arginine methyl ester in HTR-8SV/neo cells. CONCLUSION: Decreases in the ADM2 system in PreE at term, in AF from pregnant women during the second trimester who develop PreE later in gestation, and ADM2-induced increases in the NO and MMP-2/9 levels in trophoblast cells suggest a potential role for ADM2 via the NO-MMP system in the pathophysiology of PreE.

Impaired Vasodilatory Responses of Omental Arteries to CGRP Family Peptides in Pregnancies Complicated by Fetal Growth Restriction.

RATIONALE: Calcitonin gene-related peptide (CGRP), adrenomedullin (ADM), and adrenomedullin2 (ADM2)/intermedin are potent vasorelaxant peptides considered to play a role in the adaptive mechanisms in rat pregnancy through increased vasodilation in mesenteric and uterine artery. OBJECTIVE: This study was designed to demonstrate the response of omental arteries (OA) to vasoactive peptides CGRP, ADM, and ADM2 in pregnancy complications such as fetal growth restriction (FGR), and assess the changes in the expression of their receptor components in segments of OA from FGR pregnancy compared to the control. FINDINGS: The findings for this study are: 1) relaxation responses of OA were higher for bradykinin (78.55 ± 3.91 vs 52.67 ± 2.19; P < .05) in pregnancy with FGR compared to the normal, 2) relaxation response of OA segments to CGRP was similar with no change in the expression of G-protein couple receptor-calcitonin receptor-like receptor complex in normal healthy pregnancy and pregnancy complicated by FGR, 3) maximal relaxation response of OA were significantly (P < .05) lower for both ADM (18.2 ± 6.7 vs 38 ± 2.5) and ADM2 (26.9 ± 6.7 vs 48 ± 2.6) along with decreases in their respective ligand-receptor complex in FGR compared to the normal pregnancies, 4) expression of calcitonin receptor-like receptor mRNA was higher but its immunoreactivity was lower in OA from FGR pregnancy compared to the normal, and 5) mRNA and protein levels of RAMP1, RAMP2, and RAMP3 were lower in OA isolated from FGR pregnancies compared to the normal. CONCLUSION: The current study demonstrates that FGR is associated with an increase in the sensitivity of OA to bradykinin and decreased sensitivity for ADM and ADM2 ligand-receptor system with no change in the response for CGRP compared to the normal healthy pregnancy, and suggests a potential role for ADM and ADM2 in the pathophysiology of maternal vasculature in FGR pregnancy.

Novel lean type 2 diabetic rat model using gestational low-protein programming.

BACKGROUND: Type 2 diabetes (T2D) in lean individuals is not well studied and up to 26% of diabetes occurs in these individuals. Although the cause is not well understood, it has been primarily attributed to nutritional issues during early development. OBJECTIVE: Our objective was to develop a lean T2D model using gestational low-protein (LP) programming. STUDY DESIGN: Pregnant rats were fed control (20% protein) or isocaloric LP (6%) diet from gestational day 4 until delivery. Standard diet was given to dams after delivery and to pups after weaning. Glucose tolerance test was done at 2, 4, and 6 months of age. Magnetic resonance imaging of body fat for females was done at 4 months. Rats were sacrificed at 4 and 8 months of age and their perigonadal, perirenal, inguinal, and brown fat were weighed and expressed relative to their body weight. Euglycemic-hyperinsulinemic clamp was done around 6 months of age. RESULTS: Male and female offspring exposed to a LP diet during gestation developed glucose intolerance and insulin resistance (IR). Further, glucose intolerance progressed with increasing age and occurred earlier and was more severe in females when compared to males. Euglycemic-hyperinsulinemic clamp showed whole body IR in both sexes, with females demonstrating increased IR compared to males. LP females showed a 4.5-fold increase in IR while males showed a 2.5-fold increase when compared to their respective controls. Data from magnetic resonance imaging on female offspring showed no difference in the subcutaneous, inguinal, and visceral fat content. We were able to validate this observation by sacrificing the rats at 4 and 8 months and measuring total body fat content. This showed no differences in body fat content between control and LP offspring in either males or females. Additionally, diabetic rats had a similar body mass index to that of the controls. CONCLUSION: LP gestational programming produces a progressively worsening T2D model in rats with a lean phenotype without obesity.

Pregnancy Increases Relaxation in Human Omental Arteries to the CGRP Family of Peptides.

Calcitonin gene-related peptide (CALCB) and its family members adrenomedullin (ADM) and intermedin (ADM2) play important roles in maintaining vascular adaptations during pregnancy in animal models. The present study was designed to evaluate the responses of omental arteries to CALCB, ADM, and ADM2 in pregnant and nonpregnant women, and to determine the mechanisms involved. By using resistance omental arteries collected from nonpregnant women (n = 15) during laparotomy and from term pregnant women (n = 15) at cesarean delivery, this study shows that the receptor components--calcitonin receptor-like receptor (CALCRL) and receptor activity-modifying proteins (RAMPs) 1, 2 and 3--are localized to endothelial and smooth muscle cells in omental arteries, with increased expressions of both mRNA and protein in pregnant compared with nonpregnant women. The myography study demonstrated that CALCB, ADM, and ADM2 (0.1-100 nM) dose dependently relax U46619 (1 muM) precontracted omental artery segments, and the maximum possible effects to CALCB and ADM2, but not to ADM, are significantly enhanced in pregnant compared with nonpregnant women. Further, the vasodilatory responses to CALCB, ADM, and ADM2 are reduced by inhibitors of nitric oxide (NO) synthase (L-NAME), adenylyl cyclase (SQ22536), voltage-activated potassium channels (4-aminopyrodin and tetrabutylammonium), Ca(2+)-activated potassium channel (charybdotoxin), and cyclooxygenase (indomethacin). In conclusion, the CALCB family of peptides, CALCB and ADM2, increase human omental artery relaxation during pregnancy through diverse mechanisms, including NO, endothelium-derived hyperpolarizing factors (EDHFs) and prostaglandins, and thus could contribute to the vascular adaptations during pregnancy in the human.

Adrenomedullin 2 (ADM2) Regulates Mucin 1 at the Maternal-Fetal Interface in Human Pregnancy.

Association of an altered expression of placental mucin 1 (MUC1) with first-trimester spontaneous abortion and its regulation in placenta by an invasion-promoting peptide, adrenomedullin 2 (ADM2), is not known. The objective of this study was to assess 1) the association of MUC1 mRNA expression in the placental villi and decidua with first-trimester spontaneous abortion, 2) the effects of ADM2 on the expression of MUC1 in trophoblast cells in the presence or absence of hypoxia, 3) the effects of ADM2 on expression of MUC1 in decidual stromal cells (DSCs), and 4) if ADM2 regulates the expression of MUC1 and MMP2 protein in trophoblastic spheroids. Data demonstrate that 1) expression of MUC1 mRNA in villous tissue is higher in spontaneous abortion compared to age-matched electively terminated pregnancies (P > 0.05), 2) ADM2 decreases the expression of MUC1 mRNA and protein in trophoblast cells and spheroids with concomitant increases in MMP2 immunoreactivity in the spheroids, 3) ADM2 decreases hypoxia-induced increases in MUC1 immunoreactivity in trophoblast cells, 4) decidual MUC1 mRNA expression is lower in spontaneous compared to elective abortions (P < 0.05), and 5) DSCs express MUC1 mRNA and protein and ADM2 decreases the expression of MUC1 mRNA and protein in DSCs. Taken together, this study demonstrates that first-trimester spontaneous abortion is associated with increases in MUC1 expression in villi and decreases in the decidual tissues, and suggests that ADM2 may contribute to the physiology of embryo implantation and placental growth via increasing MMP2 and decreasing MUC1 expression to facilitate trophoblast invasion.

Appetite regulation is independent of the changes in ghrelin levels in pregnant rats fed low-protein diet.

Gestational protein restriction causes hypertension in the adult offspring. Very little is known about the food intake regulation and ghrelin signaling in pregnant dams fed a low-protein (LP) diet. We hypothesized that diet intake and ghrelin signaling are altered in pregnant rats fed the low-protein diet. Sprague-Dawley rats were fed a control (CT) or LP diet from Day 3 of pregnancy. Diet intake and body weight were monitored daily. Expression of ghrelin production-related genes in the stomach and appetite-related genes in the hypothalamus was analyzed by real-time PCR. Plasma levels of total and active ghrelin, growth hormone and leptin were measured by ELISA. Main results include: (1) Daily diet intake was greater in the LP group than in the CT group in early pregnancy, but substantially lower in late pregnancy; (2) Daily gain in body weight was substantially lower in the LP group in late pregnancy; (3) Expression of ghrelin production-related genes in the stomach and plasma total ghrelin levels were increased in LP group in late pregnancy; (4) Plasma active ghrelin levels were elevated in the LP group at mid-late pregnancy, but growth hormone and leptin levels were uncorrelated with active ghrelin in late pregnancy; and (5) Hypothalamic expression of ghrelin-stimulated genes in LP rats was unassociated with the changes in both plasma ghrelin levels and the diet intake. Taken together, the appetite in LP rats is greater in early pregnancy but reduced at late pregnancy, possibly due to ghrelin insensitivity in appetite regulation.

Enhanced mesenteric arterial responsiveness to angiotensin II is androgen receptor-dependent in prenatally protein-restricted adult female rat offspring.

Gestational protein restriction results in intrauterine growth restriction and hypertension in adult female growth-restricted rats. Enhanced vascular responsiveness to angiotensin II is observed, and blockade of the renin-angiotensin system abolishes hypertension in adult growth-restricted rats, suggesting that the renin-angiotensin system contributes to intrauterine growth restriction-induced hypertension. Moreover, growth-restricted adult rats have higher plasma testosterone levels, and antiandrogen treatment abolishes hypertension, indicating an important role for testosterone. We hypothesized that androgens may play a pivotal role in the enhanced responsiveness to Ang II and hypertension. Female offspring of pregnant rats fed 20% protein (control) or 6% protein diet (protein restricted), at 6 mo of age, were studied. Plasma testosterone and mean arterial pressure in protein-restricted offspring were significantly higher compared to controls. Flutamide treatment (10 mg/kg/day subcutaneously for 10 days) reduced mean arterial pressure in protein-restricted offspring but was without significant effect in controls. Vascular Agtr1/Agtr2 ratio was significantly higher in protein-restricted offspring, an effect that was reversed by flutamide. Flutamide treatment did not have any effect on Agtr1/Agtr2 ratio in controls. Enhanced contractile response to angiotensin II in mesenteric arteries was observed in protein-restricted offspring compared with control. Flutamide treatment reversed the enhanced contractile response to angiotensin II in protein-restricted offspring without significant effect in controls. Vascular reactivity to phenylephrine was similar between the control and protein-restricted offspring with and without flutamide treatment, suggesting that enhanced contractile response and flutamide's reversal effect is specific to angiotensin II. These results suggest that prenatally protein-restricted rats exhibit an enhanced responsiveness to angiotensin II that is testosterone-dependent.

Adrenomedullin2 (ADM2)/intermedin (IMD) in rat ovary: changes in estrous cycle and pregnancy and its role in ovulation and steroidogenesis.

Adrenomedullin2 (ADM2) is reported to facilitate embryo implantation and placental development. Therefore, the current study was undertaken to identify if ADM2 has a functional role in ovary to facilitate its reproductive actions. This study shows that the expression of ADM2 is differentially regulated in rat estrous cycle and that ADM2 increases the synthesis and secretion of 17beta-estradiol accompanied with an increase in the expression of steroidogenic factor 1 (Sf1), estrogen receptor Esr1, and enzymes involved in steroidogenesis in equine chorionic gonadotropin (eCG)-treated rat ovaries. In addition, inhibition of endogenous ADM2 function in eCG-treated immature rats caused impaired ovulation. Furthermore, the mRNA expression of Adm2 and receptor activity modifying protein 3 is higher in the ovary on Day 18 compared to nonpregnant and pregnant rats on Day 22. ADM2-like immunoreactivity is localized in granulosa cells, blood vessels, oocytes, cumulous oophorus, and corpus luteum of pregnant ovaries, suggesting a potential role for ADM2 in the ovary. This is supported by the presence of ADM2-like immunoreactivity in the corpus luteum during pregnancy and a decline in aromatase immunoreactivity in corpus luteum on Day 9 of gestation in rats infused with ADM2 antagonist during implantation and decidualization phase. Taken together, this study suggests a potential involvement of ADM2 in the rat ovary in regulating synthesis of estradiol to support ovulation and facilitate efficient implantation and placental development for a successful pregnancy.

Functional and molecular characterization of a lipopeptide surfactant from the marine sponge-associated eubacteria Bacillus licheniformis NIOT-AMKV06 of Andaman and Nicobar Islands, India.

The production of a lipopeptide surfactant from the sponge-associated eubacteria Bacillus licheniformis NIOT-AMKV06 from the Andaman and Nicobar Islands was investigated. The highest production was attained with glucose and yeast extracts as the carbon and nitrogen sources (1.789 mg mL(-1)), respectively. The surfactant was highly stable over a pH range of 5.0-10 and a temperature range of 20-70°C with high NaCl concentrations. Excellent emulsification activity was exhibited by the purified surfactant with crude oil, kerosene, and diesel. A two-fold increase in surfactant production (3.0 mg mL(-1)) was observed using the newly formulated medium in this study. The surfactant biosynthesis gene cluster (sfp, sfpO, and srfA) from B. licheniformis NIOT-AMKV06 was heterologously expressed in Escherichia coli, and the production was increased three-fold (11.78 g L(-1)) over the original strain. The results confirm the potential of the surfactant for use in bioremediation of hydrocarbons in a marine environment and for enhanced oil recovery. To our knowledge, this is the first report on the ability of a hydrocarbon degrading B. licheniformis from marine sponges for the biosynthesis of a potent lipopeptide surfactant possessing characteristics of maximum stability, outstanding surfactant activity, and exceptional emulsifying capability.