Omentin-1 is decreased in maternal plasma, placenta and adipose tissue of women with pre-existing obesity

Objective

The aim of this study was to determine (i) the effect of maternal obesity and gestational diabetes mellitus (GDM) on (i) the circulating levels of omentin-1 in cord and maternal plasma, and (ii) gene expression and release of omentin-1 from human placenta and adipose tissue. The effect of pregnancy on circulating omentin-1 levels was also determined.

Design

Omentin-1 levels were measured in maternal and cord plasma from obese and non-obese normal glucose tolerant women (NGT; n = 44) and women with GDM (n = 39) at the time of term elective Caesarean section. Placenta and adipose tissue expression and release of omentin-1 was measured from 22 NGT and 22 GDM women collected at the time of term elective Caesarean section. Omentin-1 levels were also measured in maternal plasma from 13 NGT women at 11 and 28 weeks gestation and 7 weeks postpartum.

Results

Maternal obesity was associated with significantly lower omentin-1 levels in maternal plasma; however, there was no effect of maternal obesity on cord omentin levels. Omentin-1 gene expression was lower in placenta and adipose tissue obtained from women with pre-existing obesity. In addition to this, adipose tissue release of omentin-1 was significantly lower from obese pregnant women. Omentin-1 levels were significantly lower in non-obese GDM compared to non-obese NGT women. However, there was no difference in omentin-1 levels between obese NGT and obese GDM women. There was no effect of GDM on cord omentin levels, and placental and adipose tissue omentin-1 expression. Maternal omentin-1 levels were negatively correlated with fetal birthweight and fetal ponderal index.

Conclusions

The data presented in this study demonstrate that pre-existing maternal obesity is associated with lower omentin-1 expression in placenta, adipose tissue and maternal plasma. Alteration in omentin-1 in pregnancy may influence the development of metabolic disorders in offspring later in life.     

Recombinant human arginase toxicity in mice is reduced by citrulline supplementation

Human recombinant arginase I cobalt coupled to polyethylene glycol 5000 (HuArg I [Co]-PEG5000) achieved potent in vitro depletion of arginine from tissue culture medium and cytotoxicity to many cancer cell lines. The recombinant enzyme also produced tumor growth inhibition of hepatocellular carcinoma and pancreatic carcinoma xenografts. Although these results were promising, the therapeutic index was narrow. Toxicities were seen in normal cells in tissue culture. In vivo normal tissue injury occurred at doses twice the effective dose. The current study was conducted to define, in greater detail, the maximum tolerated dose (MTD), pharmacodynamics, and dose-limiting toxicities (DLTs) of twice-weekly intraperitoneal HuArg I [Co]-PEG5000 in Balb/c mice. Animal weight and survival were monitored, serum arginine levels measured, and complete blood cell counts, chemistries, necropsies, and histologies were performed. In addition, methods to ameliorate the HuArg I [Co]-PEG5000 adverse effects were tested. Supplemental l-citrulline was given concurrently with the arginase drug. The HuArg I [Co]-PEG5000 MTD in mice was 5 mg/kg twice weekly, and DLTs included weight loss and marrow necrosis. No other organ damage or changes in blood cell counts or chemistries were observed. Arginase reduced serum arginine levels from 60 μM to 4 to 6 μM. Supplemental l-citrulline given per os or daily subcutaneously reduced and delayed toxicities, and l-citrulline given twice daily subcutaneously completely prevented animal toxicities. On the basis of these results, we hypothesize that HuArg I [Co]-PEG5000, particularly with supplemental l-citrulline, may be an attractive therapeutic agent for argininosuccinate synthetase-deficient tumors.     

Epstein-barr virus immortalization of human B-cells leads to stabilization of hypoxia-induced factor 1 alpha, congruent with the warburg effect

Background

Epstein-Barr virus (EBV) encodes six nuclear transformation-associated proteins that induce extensive changes in cellular gene expression and signaling and induce B-cell transformation. The role of HIF1A in EBV-induced B-cell immortalization has not been previously studied.

Methods and Findings

Using Western blotting and Q-PCR, we found that HIF1A protein is stabilized in EBV-transformed lymphoblastoid cells. Western blotting, GST pulldown assays, and immunoprecipitation showed that EBV-encoded nuclear antigens EBNA-5 and EBNA-3 bind to prolylhydroxylases 1 and 2, respectively, thus inhibiting HIF1A hydroxylation and degradation. Immunostaining and Q-PCR showed that the stabilized HIF1A translocates to the nucleus, forms a heterodimer with ARNT, and transactivates several genes involved in aerobic glycolysis. Using biochemical assays and Q-PCR, we also found that lymphoblastoid cells produce high levels of lactate, lactate dehydrogenase and pyruvate.

Conclusions

Our data suggest that activation of the aerobic glycolytic pathway, corresponding to the Warburg effect, occurs in EBV-transformed lymphoblastoid cells, in contrast to mitogen-activated B-cells.     

Multi-copy genes that enhance the yield of mammalian G protein-coupled receptors in Escherichia coli

Low yields of recombinant expression represent a major barrier to the physical characterization of membrane proteins. Here, we have identified genes that globally enhance the production of properly folded G protein-coupled receptors (GPCRs) in Escherichia coli. Libraries of bacterial chromosomal fragments were screened using two separate systems that monitor: (i) elevated fluorescence conferred by enhanced expression of GPCR-GFP fusions and (ii) increased binding of fluorescent ligand in cells producing more active receptor. Three multi-copy hits were isolated by both methods: nagD, encoding the ribonucleotide phosphatase NagD; a fragment of nlpD, encoding a truncation of the predicted lipoprotein NlpD, and the three-gene cluster ptsN-yhbJ-npr, encoding three proteins of the nitrogen phosphotransferase system. Expression of these genes resulted in a 3- to 10-fold increase in the yields of different mammalian GPCRs. Our data is consistent with the hypothesis that the expression of these genes may serve to maintain the integrity of the bacterial periplasm and to provide a favorable environment for proper membrane protein folding, possibly by inducing a fine-tuned stress response and/or via modifying the composition of the bacterial cell envelope.     

The importance of ERα and ERβ gene polymorphisms in PCOS

Estrogens act through binding to estrogen receptor α (ERα) and β (ERβ). Studies in knockout mice have shown that the absence of ERα leads to the polycystic ovary syndrome (PCOS) phenotype. Furthermore, the expression of ERβ gene is lower in follicles derived from women with PCOS compared with healthy women. The aim of this study was to investigate the importance of ERα and ERβ gene polymorphisms in PCOS. A cohort of 180 women with PCOS and 140 healthy controls were recruited, and the PvuII and XbaI polymorphisms of ERα, as well as, the AluI and RsaI polymorphisms of ERβ were genotyped. No difference was found in the distribution of these polymorphisms between patients and healthy controls. However, in PCOS women, carriers of TC and TT genotypes of PvuII polymorphism had lower fasting glucose to insulin ratio compared with carriers of CC genotype (p = 0.029). In addition, the presence of AA genotype of XbaI polymorphism was associated with lower levels of follicle-stimulating hormone (FSH) compared with the presence of AG and GG genotypes (p = 0.03). The association of ERα polymorphisms with insulin resistance indices and FSH levels emphasizes the importance of ERα as a genetic modifier of the PCOS phenotype.     

AICAR prevents heat-induced sudden death in RyR1 mutant mice independent of AMPK activation

Mice with a knock-in mutation (Y524S) in the type I ryanodine receptor (Ryr1), a mutation analogous to the Y522S mutation that is associated with malignant hyperthermia in humans, die when exposed to short periods of temperature elevation (≥37 °C). We show here that treatment with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) prevents this heat-induced sudden death in this mouse model. The protection by AICAR is independent of AMP-activated protein kinase (AMPK) activation and results from a newly identified action of the compound on mutant Ryr1 to reduce Ca(2+) leak from the sarcoplasmic reticulum to the sarcoplasm. AICAR thus prevents Ca(2+)-dependent increases in the amount of both reactive oxygen species (ROS) and reactive nitrogen species (RNS) that act to further increase resting Ca(2+) concentrations. If unchecked, the temperature-driven increases in resting Ca(2+) concentrations and the amounts of ROS and RNS create an amplifying cycle that ultimately triggers sustained muscle contractions, rhabdomyolysis and death. Although antioxidants are effective in reducing this cycle in vitro, only AICAR prevents heat-induced death in vivo. Our findings suggest that AICAR is probably effective in prophylactic treatment of humans with enhanced susceptibility to exercise- and/or heat-induced sudden death associated with RYR1 mutations.     

The evolution of catalytic efficiency and substrate promiscuity in human theta class 1-1 glutathione transferase

Theta class glutathione transferases (GST) from various species exhibit markedly different catalytic activities in conjugating the tripeptide glutathione (GSH) to a variety of electrophilic substrates. For example, the human theta 1-1 enzyme (hGSTT1-1) is 440-fold less efficient than the rat theta 2-2 enzyme (rGSTT2-2) with the fluorogenic substrate 7-amino-4-chloromethyl coumarin (CMAC). Large libraries of hGSTT1-1 constructed by error-prone PCR, DNA shuffling, or saturation mutagenesis were screened for improved catalytic activity towards CMAC in a quantitative fashion using flow cytometry. An iterative directed evolution approach employing random mutagenesis in conjunction with homologous recombination gave rise to enzymes exhibiting up to a 20,000-fold increase in k(cat)/K(M) compared to hGSTT1-1. All highly active clones encoded one or more mutations at residues 32, 176, or 234. Combinatorial saturation mutagenesis was used to evaluate the full complement of natural amino acids at these positions, and resulted in the isolation of enzymes with catalytic rates comparable to those exhibited by the fastest mutants obtained via directed evolution. The substrate selectivities of enzymes resulting from random mutagenesis, DNA shuffling, and combinatorial saturation mutagenesis were evaluated using a series of distinct electrophiles. The results revealed that promiscuous substrate activities arose in a stochastic manner, as they did not correlate with catalytic efficiency towards the CMAC selection substrate. In contrast, chimeric enzymes previously constructed by homology-independent recombination of hGSTT-1 and rGSTT2-2 exhibited very different substrate promiscuity profiles, and showed a more defined relationship between evolved and promiscuous activities.     

Oviductal cell proteome alterations during the reproductive cycle in pigs

The mammalian oviduct plays a crucial role in events leading to the establishment of pregnancy. During the reproductive cycle, the reproductive system undergoes various changes, including alterations in the number of different cell types in the oviductal epithelium and changes in the height of oviductal cells. Maintaining the unique oviductal environment required for the fertilization and early embryonic development comes with an energy cost to the organism. Therefore, it is hypothesized that structural and functional changes to the oviduct during the reproductive cycle represent vital preparations for the development of suitable environments for conception and embryo support. Here, we aimed to identify the changes in protein expression profile that occur during the follicular and luteal stages of the reproductive cycle in oviductal epithelial cells. The porcine oviductal epithelial cell proteomes from the follicular and luteal stages of the reproductive cycle were contrasted after separation by 2-D gel electrophoresis. Several oviductal epithelial cell proteins were up- or down-regulated during the reproductive cycle. We checked the quantitative changes of two of these molecules during different stages of the reproductive cycle using Western blot analysis. Finally, a number of these proteins were identified using tandem mass spectrometry. The results demonstrated distinctive differences in the proteomic profiles of the oviduct between follicular and luteal phases of the reproductive cycle.     

Superoxide radical formation in diverse organs of rats with experimentally induced obstructive jaundice

Oxidative stress seems to be a cardinal feature of cholestasis, implicated in the pathophysiology of organ injury not only in the liver, but also in several extrahepatic tissues. The present study was designed to assess directly oxidative stress in vital organs of experimentally jaundiced rats by measuring the key oxidative stress marker superoxide radical (O2(*-)). Twelve male Wistar rats underwent laparotomy and were divided into two groups - group I (n = 6) sham operated, and group II (n = 6) bile-duct ligated. Ten days later, the O2(*-) formation rate was quantified in liver, intestine, kidney and heart of all animals. These measurements were done by application of a new ultrasensitive fluorescent assay for the in vivo quantification of O2(*-), which is based on the 1:1 molar stoichiometric reaction of O2(*-) with dihydroethidine (DHE, an O2(*-) trap) that results in the formation of the specific product 2-OH-ethidium. 2-OH-Ethidium was measured by fluorescence in rats' organs and its formation rate was converted to O2(*-) production rate. As compared to sham-operated rats, in jaundiced rats there was a significant increase of O2(*-) in the intestine (136%, P < 0.01), liver (104%, P < 0.01), and kidney (95%, P < 0.01), whereas there was no significant difference in heart O2(*-) levels. Superoxide radical may play an important role in the pathophysiology of cholestatic liver injury, intestinal barrier failure and renal failure, associated with postoperative morbidity and mortality in obstructive jaundice. On the contrary, O2(*-) and oxidative stress are possibly not implicated in the pathophysiology of hepatic cardiomyopathy.