Inhibition of hypothalamic leukemia inhibitory factor exacerbates diet-induced obesity phenotype.

BACKGROUND: The consumption of large amounts of dietary fats can trigger an inflammatory response in the hypothalamus and contribute to the dysfunctional control of caloric intake and energy expenditure commonly present in obesity. The objective of this study was to identify chemokine-related transcripts that could be involved in the early stages of diet-induced hypothalamic inflammation. METHODS: We used immunoblot, PCR array, real-time PCR, immunofluorescence staining, glucose and insulin tolerance tests, and determination of general metabolic parameters to evaluate markers of inflammation, body mass variation, and glucose tolerance in mice fed a high-fat diet. RESULTS: Using a real-time PCR array, we identified leukemia inhibitory factor as a chemokine/cytokine undergoing a rapid increase in the hypothalamus of obesity-resistant and a rapid decrease in the hypothalamus of obesity-prone mice fed a high-fat diet for 1 day. We hypothesized that the increased hypothalamic expression of leukemia inhibitory factor could contribute to the protective phenotype of obesity-resistant mice. To test this hypothesis, we immunoneutralized hypothalamic leukemia inhibitory factor and evaluated inflammatory and metabolic parameters. The immunoneutralization of leukemia inhibitory factor in the hypothalamus of obesity-resistant mice resulted in increased body mass gain and increased adiposity. Body mass gain was mostly due to increased caloric intake and reduced spontaneous physical activity. This modification in the phenotype was accompanied by increased expression of inflammatory cytokines in the hypothalamus. In addition, the inhibition of hypothalamic leukemia inhibitory factor was accompanied by glucose intolerance and insulin resistance. CONCLUSION: Hypothalamic expression of leukemia inhibitory factor may protect mice from the development of diet-induced obesity; the inhibition of this protein in the hypothalamus transforms obesity-resistant into obesity-prone mice.

Paeonia lactiflora Enhances the Adhesion of Trophoblast to the Endometrium via Induction of Leukemia Inhibitory Factor Expression.

In the present study, we investigated the role of Paeonia lactiflora Pall. extract on embryo implantation in vitro and in vivo. A polysaccharides depleted-water extract of P. lactiflora (PL-PP) increased LIF expression in human endometrial Ishikawa cells at non-cytotoxic doses. PL-PP significantly increased the adhesion of the human trophectoderm-derived JAr spheroids to endometrial Ishikawa cells. PL-PP-induced LIF expression was decreased in the presence of a p38 kinase inhibitor SB203580 and an MEK/ERK inhibitor U0126. Furthermore, endometrial LIF knockdown by shRNA reduced the expression of integrins ß3 and ß5 and adhesion of JAr spheroids to Ishikawa cells. In vivo administration of PL-PP restored the implantation of mouse blastocysts in a mifepristone-induced implantation failure mice model. Our results demonstrate that PL-PP increases LIF expression via the p38 and MEK/ERK pathways and favors trophoblast adhesion to endometrial cells.

N-carbamylglutamate enhances pregnancy outcome in rats through activation of the PI3K/PKB/mTOR signaling pathway.

Administration of N-carbamylglutamate (NCG), an analogue of endogenous N-acetyl-glutamate (an activator of arginine synthesis) has been shown to enhance neonatal growth by increasing circulating arginine levels. However, the effect of NCG on pregnancy remains unknown. This study examined the effects of NCG on pregnancy outcome and evaluated potential mechanisms involved. Reproductive performance, embryo implantation, and concentration of amino acids in serum and uterine flushing, were determined in rats fed control or NCG supplemented diets. Ishikawa cells and JAR cells were used to examine the mechanism by which NCG affects embryo implantation. Dietary NCG supplementation increased serum levels of arginine, onithine, and proline, as well as uterine levels of arginine, glutamine, glutamate, and proline. Additionally, it stimulated LIF expression, and enhanced the activation of signal transduction and activator of transcription 3 (Stat3), protein kinase B (PKB), and 70-kDa ribosomal protein S6 kinase (S6K1) during the periimplantation period, resulting in an increase in litter size but not birth weight. In uterine Ishikawa cells, LIF expression was also enhanced by treatment with arginine and its metabolites. In trophoblast JAR cells, treatment with arginine and its metabolites enhanced Stat3, PKB, and S6K1 activation and facilitated cellular adhesion activity. These effects were abolished by pretreatment with inhibitors of phosphatidylinositol 3-kinase (wortmannin) and mammalian target of rapamycin (rapamycin). The results demonstrate that NCG supplementation enhances pregnancy outcome and have important implications for the pregnancy outcome of mammalian species.

Recombinant rabbit leukemia inhibitory factor and rabbit embryonic fibroblasts support the derivation and maintenance of rabbit embryonic stem cells.

The rabbit is a classical experimental animal species. A major limitation in using rabbits for biomedical research is the lack of germ-line-competent rabbit embryonic stem cells (rbESCs). We hypothesized that the use of homologous feeder cells and recombinant rabbit leukemia inhibitory factor (rbLIF) might improve the chance in deriving germ-line-competent rbES cells. In the present study, we established rabbit embryonic fibroblast (REF) feeder layers and synthesized recombinant rbLIF. We derived a total of seven putative rbESC lines, of which two lines (M5 and M23) were from culture Condition I using mouse embryonic fibroblasts (MEFs) as feeders supplemented with human LIF (hLIF) (MEF+hLIF). Another five lines (R4, R9, R15, R21, and R31) were derived from Condition II using REFs as feeder cells supplemented with rbLIF (REF+rbLIF). Similar derivation efficiency was observed between these two conditions (8.7% vs. 10.2%). In a separate experiment with 2×3 factorial design, we examined the effects of feeder cells (MEF vs. REF) and LIFs (mLIF, hLIF vs. rbLIF) on rbESC culture. Both Conditions I and II supported satisfactory rbESC culture, with similar or better population doubling time and colony-forming efficiency than other combinations of feeder cells with LIFs. Rabbit ESCs derived and maintained on both conditions displayed typical ESC characteristics, including ESC pluripotency marker expression (AP, Oct4, Sox2, Nanog, and SSEA4) and gene expression (Oct4, Sox2, Nanog, c-Myc, Klf4, and Dppa5), and the capacity to differentiate into three primary germ layers in vitro. The present work is the first attempt to establish rbESC lines using homologous feeder cells and recombinant rbLIF, by which the rbESCs were derived and maintained normally. These cell lines are unique resources and may facilitate the derivation of germ-line-competent rbESCs.

Effect of Bushenantai recipe on the expression of endometrial LIF in mice with embryonic implantation dysfunction.

In order to observe the effect of Bushenantai recipe on the expression of endometrial leukemia-inhibitory factor (LIF) in mice with embryonic implantation dysfunction (EID), 120 Kunming mice post coition were randomized into three groups: normal control group, model group and traditional Chinese medicine group (TCM group) (n=40 in each group). Uterus was collected on the pregnancy day (Pd) 4, 5, 6 after an intravenous injection of Evan's blue. The endometrium was dyed by Evan's blue and the mean points of response were observed on Pd 5. The expression of LIF mRNA and protein was detected by RT-PCR and immunohistochemistry respectively and analyzed statistically by image system. The results showed that the number of implantation sites in model group was remarkably less than in normal control group and TCM group. There was no significant difference between normal control group and TCM group. The expression of LIF mRNA and protein in model group was delayed. Bushenantai recipe could increase the expression of LIF mRNA and protein in endometria of mice with EID. It was suggested that Bushenantai recipe could improve embryo implantation of mice with EID by promoting the endometrial LIF expression and endometrial decidualization.

Effect of Bushenantai recipe on the expression of endometrial LIF in mice with embryonic implantation dysfunction / 华中科技大学学报（医学）（英德文版）

In order to observe the effect of Bushenantai recipe on the expression of endometrial leukemia-inhibitory factor (LIF) in mice with embryonic implantation dysfunction (EID), 120 Kunming mice post coition were randomized into three groups: normal control group, model group and traditional Chinese medicine group (TCM group) (n=40 in each group). Uterus was collected on the pregnancy day (Pd) 4, 5, 6 after an intravenous injection of Evan's blue. The endometrium was dyed by Evan's blue and the mean points of response were observed on Pd 5. The expression of LIF mRNA and protein was detected by RT-PCR and immunohistochemistry respectively and analyzed statistically by image system. The results showed that the number of implantation sites in model group was remarkably less than in normal control group and TCM group. There was no significant difference between normal control group and TCM group. The expression of LIF mRNA and protein in model group was delayed. Bushenantai recipe could increase the expression of LIF mRNA and protein in endometria of mice with EID. It was suggested that Bushenantai recipe could improve embryo implantation of mice with EID by promoting the endometrial LIF expression and endometrial decidualization.