Human MC4R variants affect endocytosis, trafficking and dimerization revealing multiple cellular mechanisms involved in weight regulation.

The Melanocortin-4 Receptor (MC4R) plays a pivotal role in energy homeostasis. We used human MC4R mutations associated with an increased or decreased risk of obesity to dissect mechanisms that regulate MC4R function. Most obesity-associated mutations impair trafficking to the plasma membrane (PM), whereas obesity-protecting mutations either accelerate recycling to the PM or decrease internalization, resulting in enhanced signaling. MC4R mutations that do not affect canonical Gαs protein-mediated signaling, previously considered to be non-pathogenic, nonetheless disrupt agonist-induced internalization, ß-arrestin recruitment, and/or coupling to Gαs, establishing their causal role in severe obesity. Structural mapping reveals ligand-accessible sites by which MC4R couples to effectors and residues involved in the homodimerization of MC4R, which is disrupted by multiple obesity-associated mutations. Human genetic studies reveal that endocytosis, intracellular trafficking, and homodimerization regulate MC4R function to a level that is physiologically relevant, supporting the development of chaperones, agonists, and allosteric modulators of MC4R for weight loss therapy.

Myeloperoxidase in human peripheral blood lymphocytes: Production and subcellular localization.

Myeloperoxidase (MPO) is an important enzyme in the front-line protection against microorganisms. In peripheral blood, it is accepted that MPO is only produced by myeloid-lineage cells. Thus, MPO presence is unexpected in lymphocytes. We showed recently that B1-lymphocytes from mice have MPO. Here, we showed that subsets of human peripheral B, CD4(+) and CD8(+) T lymphocytes express MPO. The content of MPO in lymphocytes was very low compared to neutrophils/monocytes with a preferential distribution in the nucleus and perinuclear region. Also, we performed a MPO mRNA expression analysis from human blood cells derived from microarray raw data publicly available, showing that MPO is modulated in infectious disease. MPO was increased in CD4(+) T lymphocytes from HIV chronic infection and in CD8(+) T lymphocytes from HCV-positive patients. Our study points out MPO as a multifunctional protein due to its subcellular localization and expression modulation in lymphocytes indicating alternative unknown functions for MPO in lymphocytes.

Late effects of sleep restriction: Potentiating weight gain and insulin resistance arising from a high-fat diet in mice.

OBJECTIVE: Epidemiological studies show the association of sleep restriction (SR) with obesity and insulin resistance. Experimental studies are limited to the concurrent or short-term effects of SR. Here, we examined the late effects of SR regarding weight gain and metabolic alterations induced by a high-fat diet (HFD). METHODS: C57BL/6 mice were subjected to a multiple platform method of SR for 15 days, 21 h daily, followed by 6 weeks of a 30% HFD. RESULTS: Just after SR, serum insulin and resistin concentrations were increased and glycerol content decreased. In addition, resistin, TNF-α, and IL-6 mRNA expression were notably increased in epididymal fat. At the end of the HFD period, mice previously submitted to SR gained more weight (32.3 ± 1.0 vs. 29.4 ± 0.7 g) with increased subcutaneous fat mass, had increments in the expression of the adipogenic genes PPARγ, C/EBPα, and C/EBPß, and had macrophage infiltration in the epididymal adipose tissue. Furthermore, enhanced glucose tolerance and insulin resistance were also observed. CONCLUSIONS: The consequences of SR may last for a long period, characterizing SR as a predisposing factor for weight gain and insulin resistance. Metabolic changes during SR seem to prime adipose tissue, aggravating the harmful effects of diet-induced obesity.

Serum amyloid A in the placenta and its role in trophoblast invasion.

The serum amyloid A (SAA) protein is known to function in the acute phase response and immunoregulation. Recently, SAA has been shown to be involved in cell proliferation, differentiation and migratory behavior in different cell types. Here, we evaluated whether exogenous SAA could influence trophoblast invasion and differentiation using both the trophoblast-like BeWo cell line and fully differentiated human extravillous trophoblast cells (EVT) isolated from term placentae. SAA stimulated BeWo cell invasion, as measured in Matrigel invasion assays, and induced metalloprotease mRNA expression and activity. Given that BeWo cells express Toll-like receptor 4 (TLR4), a known receptor for SAA, we examined the role of TLR4 in SAA-induced invasion using a TLR4 neutralizing antibody. We also tested whether SAA could affect markers of trophoblast syncytialization in BeWo cells. We observed that SAA decreased ßhCG secretion and did not influence trophoblast syncytialization. Using EVT cells isolated from human term basal plates, we confirmed that SAA at 1 and 10 µg/mL doubled EVT invasion in a TLR4-dependent manner, but at 20 µg/mL inhibited EVT cells invasiveness. In addition, we observed that SAA was expressed in both BeWo cells and human term placentae, specifically in the syncytiotrophoblast, decidual cells and EVT. In conclusion, SAA was identified as a molecule that functions in the placental microenvironment to regulate metalloprotease activity and trophoblast invasion, which are key processes in placentation and placental homeostasis.

Fibroblasts protect melanoma cells from the cytotoxic effects of doxorubicin.

Melanoma is the most aggressive form of skin cancer and until recently, it was extremely resistant to radio-, immuno-, and chemotherapy. Despite the latest success of BRAF V600E-targeted therapies, responses are typically short lived and relapse is all but certain. Furthermore, a percentage (40%) of melanoma cells is BRAF wild type. Emerging evidence suggests a role for normal host cells in the occurrence of drug resistance. In the current study, we compared a variety of cell culture models with an organotypic incomplete skin culture model (the "dermal equivalent") to investigate the role of the tissue microenvironment in the response of melanoma cells to the chemotherapeutic agent doxorubicin (Dox). In the dermal equivalent model, consisting of fibroblasts embedded in type I collagen matrix, melanoma cells showed a decreased cytotoxic response when compared with less complex culture conditions, such as seeding on plastic cell culture plate (as monolayers cultures) or on collagen gel. We further investigated the role of the microenvironment in p53 induction and caspase 3 and 9 cleavage. Melanoma cell lines cultured on dermal equivalent showed decreased expression of p53 after Dox treatment, and this outcome was accompanied by induction of interleukin IL-6, IL-8, and matrix metalloproteinases 2 and 9. Here, we show that the growth of melanoma cells in the dermal equivalent model inflects drug responses by recapitulating important pro-survival features of the tumor microenvironment. These studies indicate that the presence of stroma enhances the drug resistance of melanoma in vitro, more closely mirroring the in vivo phenotype. Our data, thus, demonstrate the utility of organotypic cell culture models in providing essential context-dependent information critical for the development of new therapeutic strategies for melanoma. We believe that the organotypic model represents an improved screening platform to investigate novel anti-cancer agents, as it provides important insights into tumor-stromal interactions, thus assisting in the elucidation of chemoresistance mechanisms.

Cytotoxicity of PVPAC-treated bovine pericardium: a potential replacement for glutaraldehyde in biological heart valves.

Acellular biological tissues, including bovine pericardium (BP), have been proposed as biomaterial for tissue engineering. BP is usually modified chemically to improve mechanical and biological properties using glutaraldehyde, the standard reagent for preservation of fresh bioprosthetic materials. Glutaraldehyde-fixed BP (Glut-BP), the most widely used material in heart valve manufacture, has been associated with calcification in vivo. In an attempt to reduce this issue and maintain its biocompatibility, this study assesses the physical properties and cytotoxicity of lyophilized BP treated with poly (vinylpyrrolidone-co-acrolein) (PVPAC-BP), a novel copolymer, as a substitute for glutaraldehyde. For that, PVPAC-BP surface ultrastructure, elastic function, water uptake and tissue calcification were evaluated. For the analysis of biocompatibility, fibroblasts (3T3-L1) and endothelial cells (HUVEC) were cultured on PVPAC-BP, Untreated-BP and Glut-BP. Nitric oxide (NO) release assay, fluorescence and SEM images of endothelial cells adhered on scaffolds were also performed. As results, the data show some advantages of PVPAC-BP over the Glut-BP. The PVPAC-BP maintains partially the original ultrastructure and elastic properties, improves scaffold hydration, and presents less calcium phosphate deposits. The cells demonstrated strong attachment, high proliferation rate, and formation of a monolayer on PVPAC-BP. Attached cells were also able to release NO de-monstrating regular metabolism. In conclusion, PVPAC may be considered as a promising alternative to BP treatment improving the efficiency of cell attachment and proliferation and also avoid immunogenicity.

Tryptamine and dimethyltryptamine inhibit indoleamine 2,3 dioxygenase and increase the tumor-reactive effect of peripheral blood mononuclear cells.

Indoleamine 2,3-dioxygenase (IDO) is an interferon-γ (IFN-γ)-induced tryptophan-degrading enzyme, producing kynurenine (KYN) that participates in the mechanism of tumor immune tolerance. Thus, IDO inhibition has been considered a strategy for anticancer therapy. The aim of this study was to identify whether the metabolites originated from the competitive routes of tryptophan metabolism, such as the serotonergic or N, N-dimethyltryptamine (DMT) pathways, have inhibitory effects on recombinant human IDO (rhIDO) activity. Serotonin and melatonin had no effect; on the other hand, tryptamine (TRY) and DMT modulated the activity of rhIDO as classical non-competitive inhibitors, with Ki values of 156 and 506 µM, respectively. This inhibitory effect was also observed on constitutively expressed or IFN-γ-induced IDO in the A172 human glioma cell line. TRY and DMT increased the cytotoxic activity of peripheral blood mononuclear cells (PBMCs) in co-culture assays. We conclude that the IDO inhibition by TRY and DMT contributed to a more effective tumor-reactive response by the PBMCs.

Hypoxia increases serum amyloid A3 (SAA3) in differentiated 3T3-L1 adipocytes.

Hypoxia has been implicated as a possible cause of adipose tissue inflammation. Furthermore, the acute phase protein serum amyloid A (SAA) has been associated with the modulation of the adipogenic process, and it is well-known that obese individuals have increased levels of SAA. The effect of hypoxia in the expression and production of SAA was examined in murine 3T3-L1 adipocytes. Hypoxia leads to a substantial increase in SAA3 mRNA and protein level, apparently in a time-dependent manner (threefold in 48 h), in fully differentiated 3T3-L1, followed by reestablishment of gene expression to basal levels after 24 h of reoxygenation. Hypoxia-induced SAA may be one of the key molecules to the development of the inflammatory response in adipose tissue.