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SUMMARY Melanocortin 4 receptors (Mc4rs) are expressed by extra-hypothalamic neurons including cholinergic autonomic pre-ganglionic neurons. However, whether Mc4rs in these neurons are required to control energy and glucose homeostasis is unclear. Here we report that Mc4rs in sympathetic, but not parasympathetic, pre-ganglionic neurons are required to regulate energy expenditure and body weight including brown and white adipose tissue thermogenic responses to diet and cold exposure. In addition, deletion of Mc4rs in both sympathetic and parasympathetic cholinergic neurons impairs glucose homeostasis.

Intestinal barrier function is achieved primarily through regulating the synthesis of mucins and tight junction (TJ) proteins, which are critical for maintaining optimal gut health and animal performance. An aberrant expression of TJ proteins results in increased paracellular permeability, leading to intestinal and systemic disorders. As an essential component of innate immunity, host defense peptides (HDPs) play a critical role in mucosal defense. Besides broad-spectrum antimicrobial activities, HDPs promotes inflammation resolution, endotoxin neutralization, wound healing, and the development of adaptive immune response. Accumulating evidence has also indicated an emerging role of HDPs in barrier function and intestinal homeostasis. HDP deficiency in the intestinal tract is associated with barrier dysfunction and dysbiosis. Several HDPs were recently shown to enhance mucosal barrier function by directly inducing the expression of multiple mucins and TJ proteins. Consistently, dietary supplementation of HDPs often leads to an improvement in intestinal morphology, production performance, and feed efficiency in livestock animals. This review summarizes current advances on the regulation of epithelial integrity and homeostasis by HDPs. Major signaling pathways mediating HDP-induced mucin and TJ protein synthesis are also discussed. As an alternative strategy to antibiotics, supplementation of exogenous HDPs or modulation of endogenous HDP synthesis may have potential to improve intestinal barrier function and animal health and productivity.

The molecular mechanisms underlying acute leptin and serotonin 2C receptor induced hypophagia remain unclear. Here we show that neuronal and pro-opiomelanocortin (Pomc)-specific loss of transient receptor potential cation 5 (TrpC5) subunits is sufficient to decrease energy expenditure and increase food intake resulting in elevated body weight. Deficiency of Trpc5 subunits in Pomc neurons was also sufficient to block the anorexigenic effects of leptin and serotonin 2C receptor (Ht2Cr) agonists. The loss of acute anorexigenic effects of these receptors was concomitant with a blunted electrophysiological response to both leptin and Ht2Cr agonists in arcuate Pomc neurons. We also demonstrate that the Ht2Cr agonist lorcaserin-induced improvements in glucose and insulin tolerance were blocked by TrpC5 deficiency in Pomc neurons. Together, our results link TrpC5 subunits in the brain with leptin- and serotonin 2C receptor-dependent changes in neuronal activity as well as energy balance, feeding behavior, and glucose metabolism.

A rise in antimicrobial resistance demands novel alternatives to antimicrobials for disease control and prevention. As an important component of innate immunity, host defense peptides (HDPs) are capable of killing a broad spectrum of pathogens and modulating a range of host immune responses. Enhancing the synthesis of endogenous HDPs has emerged as a novel host-directed antimicrobial therapeutic strategy. To facilitate the identification of natural products with a strong capacity to induce HDP synthesis, a stable macrophage cell line expressing a luciferase reporter gene driven by a 2-Kb avian β-defensin 9 (AvBD9) gene promoter was constructed through lentiviral transduction and puromycin selection. A high throughput screening assay was subsequently developed using the stable reporter cell line to screen a library of 584 natural products. A total of 21 compounds with a minimum Z-score of 2.0 were identified. Secondary screening in chicken HTC macrophages and jejunal explants further validated most compounds with a potent HDP-inducing activity in a dose-dependent manner. A follow-up oral administration of a lead natural compound, wortmannin, confirmed its capacity to enhance the AvBD9 gene expression in the duodenum of chickens. Besides AvBD9, most other chicken HDP genes were also induced by wortmannin. Additionally, butyrate was also found to synergize with wortmannin and several other newly-identified compounds in AvBD9 induction in HTC cells. Furthermore, wortmannin acted synergistically with butyrate in augmenting the antibacterial activity of chicken monocytes. Therefore, these natural HDP-inducing products may have the potential to be developed individually or in combinations as novel antibiotic alternatives for disease control and prevention in poultry and possibly other animal species including humans.

Renal cell carcinoma (RCC) is the most common type of kidney cancer, about one third of the cases are diagnosed at advanced stages with metastases and effective treatments for metastatic RCC are lacking. The molecular events supporting RCC progression remain poorly understood. SPOP, an E3 ubiquitin ligase component, was recently showed to sufficiently promote RCC tumorigenesis, however, other potential functions of SPOP in RCC have not been studied. In the present investigation, by assessing the immunohistochemical staining of SPOP in urological tumors, we found the protein was highly expressed in RCC, in particular, it was specifically expressed in clear cell RCC. cDNA microarray data showed that SPOP mRNA level was significantly increased in clear cell RCC compared to normal kidney tissues, which might be the result of the abnormal DNA copy number of this gene. More interestingly, SPOP was positive in tumors with local invasion or metastasis, and it was associated with tumor recurrence-free survival of clear cell RCC patients. Further in vitro assays demonstrated that SPOP drove RCC epithelial-mesenchymal transition (EMT) and promoted cell invasion. Mechanistically, SPOP enhanced β-catenin protein expression as well as its nuclear translocation, and elevated TCF4 expression. Both β-catenin and TCF4 upregulated the critical EMT-inducing transcription factor ZEB1, which functioned as an effector of β-catenin/TCF4 signaling in RCC invasion. These data identified SPOP as a new marker and prognostic factor for clear cell RCC, and its functions provide new insight into the molecular mechanisms of RCC progression, in which SPOP appears to be an EMT activator.

BackgroundMany groups of Gram-negative bacteria cause diseases harmful to sheep. TLR4 is an important Toll-like receptor (TLR) which responds to common Gram-negative bacterial infections. Activation of TLR4 leads to the induction of inflammatory responses, which is a linkage between the innate and adaptive immune systems. A vector pTLR4-3S was constructed to overexpress TLR4 gene in sheep. In this study, effects of TLR4 overexpression on inflammation response under LPS stimulated were addressed in vivo and in vitro.Methodology/Principal FindingsSheep fetal fibroblasts were transfected with expression vector pTLR4-3S. Transgenic sheep were produced by microinjection of the constructed plasmids into fertilized eggs. Fetal fibroblasts, monocyte-macrophage and fibroblasts isolated from the transgenic sheep were stimulated by LPS. After that immunoactive factors (TNF-α, IL-10, IL-6, IL-8, IFN-γ), nitric oxide, phagocytize ability and adhesion were detected. Furthermore, transgenic sheep were intradermal injected of LPS in ear and observed pathological changes by HE strain. Overexpression of TLR4 gene was observed on transgenic cells and individuals. In vitro, TLR4 overexpression transgenic cells secreted Th1 and Th2 inducing cytokines with a strong LPS mediated inflammation response and promoting the secretion of nitric oxide, and then recovered to initial level. The phagocytosis index of monocyte/macrophage in transgenic sheep was higher than that of non-transgenic sheep (P<0.05). In vivo, tissue sections showed that transgenic individuals launched inflammation response more quickly.Conclusions/SignificanceOverexpression of TLR4 in transgenic sheep enhanced the clearance of invaded microbe through secretion of cytokines, activation of macrophage, oxidation damage and infiltration of neutrophil.

Whether neuronal inositol-requiring enzyme 1 (Ire1) is required for the proper regulation of energy balance and glucose homeostasis is unclear. We found that pro-opiomelanocortin (Pomc)–specific deficiency of Ire1α accelerated diet-induced obesity concomitant with a decrease in energy expenditure. This hypometabolic phenotype included deficits in thermogenic responses to diet and cold exposure as well as “beiging” of white adipose tissue. We also demonstrate that loss of Ire1α in Pomc neurons impaired whole-body glucose and insulin tolerance as well as hepatic insulin sensitivity. At the cellular level, deletion of Ire1α in Pomc neurons elevated hypothalamic endoplasmic reticulum (ER) stress and predisposed Pomc neurons to leptin and insulin resistance. Together, the current studies extend and confirm conclusions that Ire1α-Xbp1s and associated molecular targets link ER stress in arcuate Pomc neurons to aspects of normal energy and glucose homeostasis.