In Silico and In Vitro Mapping of Receptor-Type Protein Tyrosine Phosphatase Receptor Type D in Health and Disease: Implications for Asprosin Signalling in Endometrial Cancer and Neuroblastoma.

BACKGROUND: Protein Tyrosine Phosphatase Receptor Type D (PTPRD) is involved in the regulation of cell growth, differentiation, and oncogenic transformation, as well as in brain development. PTPRD also mediates the effects of asprosin, which is a glucogenic hormone/adipokine derived following the cleavage of the C-terminal of fibrillin 1. Since the asprosin circulating levels are elevated in certain cancers, research is now focused on the potential role of this adipokine and its receptors in cancer. As such, in this study, we investigated the expression of PTPRD in endometrial cancer (EC) and the placenta, as well as in glioblastoma (GBM). METHODS: An array of in silico tools, in vitro models, tissue microarrays (TMAs), and liquid biopsies were employed to determine the gene and protein expression of PTPRD in healthy tissues/organs and in patients with EC and GBM. RESULTS: PTPRD exhibits high expression in the occipital lobe, parietal lobe, globus pallidus, ventral thalamus, and white matter, whereas in the human placenta, it is primarily localised around the tertiary villi. PTPRD is significantly upregulated at the mRNA and protein levels in patients with EC and GBM compared to healthy controls. In patients with EC, PTPRD is significantly downregulated with obesity, whilst it is also expressed in the peripheral leukocytes. The EC TMAs revealed abundant PTPRD expression in both low- and high-grade tumours. Asprosin treatment upregulated the expression of PTPRD only in syncytialised placental cells. CONCLUSIONS: Our data indicate that PTPRD may have potential as a biomarker for malignancies such as EC and GBM, further implicating asprosin as a potential metabolic regulator in these cancers. Future studies are needed to explore the potential molecular mechanisms/signalling pathways that link PTPRD and asprosin in cancer.

The roles of DGAT1 and DGAT2 in human myotubes are dependent on donor patho-physiological background.

The roles of DGAT1 and DGAT2 in lipid metabolism and insulin responsiveness of human skeletal muscle were studied using cryosections and myotubes prepared from muscle biopsies from control, athlete, and impaired glucose regulation (IGR) cohorts of men. The previously observed increases in intramuscular triacylglycerol (IMTG) in athletes and IGR were shown to be related to an increase in lipid droplet (LD) area in type I fibers in athletes but, conversely, in type II fibers in IGR subjects. Specific inhibition of both diacylglycerol acyltransferase (DGAT) 1 and 2 decreased fatty acid (FA) uptake by myotubes, whereas only DGAT2 inhibition also decreased fatty acid oxidation. Fatty acid uptake in myotubes was negatively correlated with the lactate thresholds of the respective donors. DGAT2 inhibition lowered acetate uptake and oxidation in myotubes from all cohorts whereas DGAT1 inhibition had no effect. A positive correlation between acetate oxidation in myotubes and resting metabolic rate (RMR) from fatty acid oxidation in vivo was observed. Myotubes from athletes and IGR had higher rates of de novo lipogenesis from acetate that were normalized by DGAT2 inhibition. Moreover, DGAT2 inhibition in myotubes also resulted in increased insulin-induced Akt phosphorylation. The differential effects of DGAT1 and DGAT2 inhibition suggest that the specialized role of DGAT2 in esterifying nascent diacylglycerols and de novo synthesized FA is associated with synthesis of a pool of triacylglycerol, which upon hydrolysis results in effectors that promote mitochondrial fatty acid oxidation but decrease insulin signaling in skeletal muscle cells.

NUCB2/Nesfatin-1 Reduces Obesogenic Diet Induced Inflammation in Mice Subcutaneous White Adipose Tissue.

BACKGROUND: Excess adipose tissue accumulation and obesity are characterised by chronic, low-grade, systemic inflammation. Nestfatin-1 is a neuropeptide derived from the precursor protein nucleobindin-2 (NUCB2), which was initially reported to exert anorexigenic effects. The present study aimed to investigate the effects of an obesogenic diet (OD; high-fat, high-sugar) in NUCB2 knockout (KO) mice and of nesfatin-1 treatment in LPS-stimulated 3T3-L1 preadipocytes. METHODS: Subcutaneous white adipose tissue (Sc-WAT) samples from wild type (WT) and NUCB2 KO mice that were fed a normal diet (ND), or the OD for 12 weeks were used for RNA and protein extraction, as well as immunohistochemistry. 3T3-L1 cells were treated with 100 nM nesfatin-1 during differentiation and stimulated with 1 µg/mL LPS for measuring the expression and secretion of pro-inflammatory mediators by qPCR, western blotting, immunofluorescence, Bioplex, and ELISA. RESULTS: Following the OD, the mRNA, protein and cellular expression of pro-inflammatory mediators (Tnfα, Il-6, Il-1ß, Adgre1, Mcp1, TLR4, Hmbgb1 and NF-kB) significantly increased in the ScWAT of NUCB2 KO mice compared to ND controls. Adiponectin and Nrf2 expression significantly decreased in the ScWAT of OD-fed NUCB2 KO, without changes in the OD-fed WT mice. Furthermore, nesfatin-1 treatment in LPS-stimulated 3T3-L1 cells significantly reduced the expression and secretion of pro-inflammatory cytokines (Tnfα, Il-6, Il-1ß, Mcp1) and hmgb1. CONCLUSION: An obesogenic diet can induce significant inflammation in the ScWAT of NUCB2 KO mice, involving the HMGB1, NRF2 and NF-kB pathways, while nesfatin-1 reduces the pro-inflammatory response in LPS-stimulated 3T3-L1 cells. These findings provide a novel insight into the metabolic regulation of inflammation in WAT.

Asprosin Exerts Pro-Inflammatory Effects in THP-1 Macrophages Mediated via the Toll-like Receptor 4 (TLR4) Pathway.

Adipose tissue is a dynamic endocrine organ, secreting a plethora of adipokines which play a key role in regulating metabolic homeostasis and other physiological processes. An altered adipokine secretion profile from adipose tissue depots has been associated with obesity and related cardio-metabolic diseases. Asprosin is a recently described adipokine that is released in response to fasting and can elicit orexigenic and glucogenic effects. Circulating asprosin levels are elevated in a number of cardio-metabolic diseases, including obesity and type 2 diabetes. In vitro studies have reported pro-inflammatory effects of asprosin in a variety of tissues. The present study aimed to further elucidate the role of asprosin in inflammation by exploring its potential effect(s) in THP-1 macrophages. THP-1 monocytes were differentiated to macrophages by 48 h treatment with dihydroxyvitamin D3. Macrophages were treated with 100 nM recombinant human asprosin, 100 ng/mL lipopolysaccharide (LPS), and 10 µM caffeic acid phenethyl ester (CAPE; an inhibitor of NFκB activation) or 1 µM TAK-242 (a Toll-like receptor 4, TLR4, inhibitor). The expression and secretion of pertinent pro-inflammatory mediators were measured by qPCR, Western blot, ELISA and Bioplex. Asprosin stimulation significantly upregulated the expression and secretion of the pro-inflammatory cytokines: tumour necrosis factor α (TNFα), interleukin-1ß (IL-1ß), IL-8 and IL-12 in vitro. This pro-inflammatory response in THP-1 macrophages was partly attenuated by the treatments with CAPE and was significantly inhibited by TAK-242 treatment. Asprosin-induced inflammation is significantly counteracted by TLR4 inhibition in THP-1 macrophages, suggesting that asprosin exerts its pro-inflammatory effects, at least in part, via the TLR4 signalling pathway.

Asprosin, a novel pleiotropic adipokine implicated in fasting and obesity-related cardio-metabolic disease: Comprehensive review of preclinical and clinical evidence.

White adipose tissue is a dynamic endocrine organ that releases an array of adipokines, which play a key role in regulating metabolic homeostasis and multiple other physiological processes. An altered adipokine secretion profile from adipose tissue depots frequently characterizes obesity and related cardio-metabolic diseases. Asprosin is a recently discovered adipokine that is released in response to fasting. Following secretion, asprosin acts - via an olfactory G-protein coupled receptor and potentially via other unknown receptor(s) - on hepatocytes and agouti-related peptide-expressing neurons in the central nervous system to stimulate glucose secretion and promote appetite, respectively. A growing body of both in vitro and in vivo studies have shown asprosin to exert a number of effects on different metabolic tissues. Indeed, asprosin can attenuate insulin signalling and promote insulin resistance in skeletal muscle by increasing inflammation and endoplasmic reticulum stress. Interestingly, asprosin may also play a protective role in cardiomyocytes that are exposed to hypoxic conditions. Moreover, clinical studies have reported elevated circulating asprosin levels in obesity, type 2 diabetes and other obesity-related cardio-metabolic diseases, with significant associations to clinically relevant parameters. Understanding the spectrum of the effects of this novel adipokine is essential in order to determine its physiologic role and its significance as a potential therapeutic target and/or a biomarker of cardio-metabolic disease. The present review offers a comprehensive overview of the published literature on asprosin, including both clinical and preclinical studies, focusing on its role in metabolism and cardio-metabolic disease.

Exometabolomic Analysis of Decidualizing Human Endometrial Stromal and Perivascular Cells.

Differentiation of endometrial fibroblasts into specialized decidual cells controls embryo implantation and transforms the cycling endometrium into a semi-permanent, immune-protective matrix that accommodates the placenta throughout pregnancy. This process starts during the midluteal phase of the menstrual cycle with decidual transformation of perivascular cells (PVC) surrounding the terminal spiral arterioles and endometrial stromal cells (EnSC) underlying the luminal epithelium. Decidualization involves extensive cellular reprogramming and acquisition of a secretory phenotype, essential for coordinated placental trophoblast invasion. Secreted metabolites are an emerging class of signaling molecules, collectively known as the exometabolome. Here, we used liquid chromatography-mass spectrometry to characterize and analyze time-resolved changes in metabolite secretion (exometabolome) of primary PVC and EnSC decidualized over 8 days. PVC were isolated using positive selection of the cell surface marker SUSD2. We identified 79 annotated metabolites differentially secreted upon decidualization, including prostaglandin, sphingolipid, and hyaluronic acid metabolites. Secreted metabolites encompassed 21 metabolic pathways, most prominently glycerolipid and pyrimidine metabolism. Although temporal exometabolome changes were comparable between decidualizing PVC and EnSC, 32 metabolites were differentially secreted across the decidualization time-course. Further, targeted metabolomics demonstrated significant differences in secretion of purine pathway metabolites between decidualized PVC and EnSC. Taken together, our findings indicate that the metabolic footprints generated by different decidual subpopulations encode spatiotemporal information that may be important for optimal embryo implantation.

Regulatory microRNAs in Brown, Brite and White Adipose Tissue.

MicroRNAs (miRNAs) constitute a class of short noncoding RNAs which regulate gene expression by targeting messenger RNA, inducing translational repression and messenger RNA degradation. This regulation of gene expression by miRNAs in adipose tissue (AT) can impact on the regulation of metabolism and energy homeostasis, particularly considering the different types of adipocytes which exist in mammals, i.e., white adipocytes (white AT; WAT), brown adipocytes (brown AT; BAT), and inducible brown adipocytes in WAT (beige or brite or brown-in-white adipocytes). Indeed, an increasing number of miRNAs has been identified to regulate key signaling pathways of adipogenesis in BAT, brite AT, and WAT by acting on transcription factors that promote or inhibit adipocyte differentiation. For example, MiR-328, MiR-378, MiR-30b/c, MiR-455, MiR-32, and MiR-193b-365 activate brown adipogenesis, whereas MiR-34a, MiR-133, MiR-155, and MiR-27b are brown adipogenesis inhibitors. Given that WAT mainly stores energy as lipids, whilst BAT mainly dissipates energy as heat, clarifying the effects of miRNAs in different types of AT has recently attracted significant research interest, aiming to also develop novel miRNA-based therapies against obesity, diabetes, and other obesity-related diseases. Therefore, this review presents an up-to-date comprehensive overview of the role of key regulatory miRNAs in BAT, brite AT, and WAT.

Vascular Adhesion Protein-1 Determines the Cellular Properties of Endometrial Pericytes.

Vascular adhesion protein-1 (VAP-1) is an inflammation-inducible adhesion molecule and a primary amine oxidase involved in immune cell trafficking. Leukocyte extravasation into tissues is mediated by adhesion molecules expressed on endothelial cells and pericytes. Pericytes play a major role in the angiogenesis and vascularization of cycling endometrium. However, the functional properties of pericytes in the human endometrium are not known. Here we show that pericytes surrounding the spiral arterioles in midluteal human endometrium constitutively express VAP-1. We first characterize these pericytes and demonstrate that knockdown of VAP-1 perturbed their biophysical properties and compromised their contractile, migratory, adhesive and clonogenic capacities. Furthermore, we show that loss of VAP-1 disrupts pericyte-uterine natural killer cell interactions in vitro. Taken together, the data not only reveal that endometrial pericytes represent a cell population with distinct biophysical and functional properties but also suggest a pivotal role for VAP-1 in regulating the recruitment of innate immune cells in human endometrium. We posit that VAP-1 could serve as a potential biomarker for pregnancy pathologies caused by a compromised perivascular environment prior to conception.

Reduced GLP-1 Secretion at 30 Minutes After a 75-g Oral Glucose Load Is Observed in Gestational Diabetes Mellitus: A Prospective Cohort Study.

Glucagon-like peptide 1 (GLP-1) levels may be reduced in type 2 diabetes, but whether a similar impairment exists in gestational diabetes mellitus (GDM) has not been established. We studied this in a prospective cohort study of pregnant women (n = 144) during oral glucose tolerance test (OGTT). GLP-1, glucose, and insulin were sampled at 30-min intervals during a 2-h 75-g OGTT, and indices of insulin secretion and sensitivity were calculated. In a nested case-control study, women with GDM (n = 19) had 12% lower total GLP-1 secretion area under the curve (AUC) compared with control subjects matched for age, ethnicity, and gestational age (n = 19), selected from within the lowest quartile of glucose120 min values in our cohort. GDM had lower GLP-1 response in the first 30 min (19% lower GLP-130 min and 17% lower AUC0-30 min) after adjustment for possible confounders. Their glucose levels began to diverge at 30 min of the OGTT with increasing insulin levels, and by 120 min, their insulin levels were three times higher. In a secondary cohort of 57 women that included "high-normal" glucose120 min values, low GLP-1 AUC0-30 min was independently associated with lower indices of insulin secretion and sensitivity. In conclusion, we have observed that women with GDM have lower GLP-1 response at 30 min of an OGTT and hyperglycemia at 120 min despite significant hyperinsulinemia.

Fibroblast growth factors: new insights, new targets in the management of diabetes.

The fibroblast growth factor (FGF) family consists of 22 evolutionarily and structurally related proteins (FGF1 to FGF23; with FGF15 being the rodent ortholog of human FGF19). Based on their mechanism of action, FGFs can be categorized into intracrine, autocrine/paracrine and endocrine subgroups. Both autocrine/paracrine and endocrine FGFs are secreted from their cells of origin and exert their effects on target cells by binding to and activating specific single-pass transmembrane tyrosine kinase receptors (FGFRs). Moreover, FGF binding to FGFRs requires specific cofactors, namely heparin/heparan sulfate proteoglycans or Klothos for autocrine/paracrine and endocrine FGF signaling, respectively. FGFs are vital for embryonic development and mediate a broad spectrum of biological functions, ranging from cellular excitability to angiogenesis and tissue regeneration. Over the past decade certain FGFs (e.g. FGF1, FGF10, FGF15/FGF19 and FGF21) have been further recognized as regulators of energy homeostasis, metabolism and adipogenesis, constituting novel therapeutic targets for obesity and obesity-related cardiometabolic disease. Until recently, translational research has been mainly focused on FGF21, due to the pleiotropic, beneficial metabolic actions and the relatively benign safety profile of its engineered variants. However, increasing evidence regarding the role of additional FGFs in the regulation of metabolic homeostasis and recent developments regarding novel, engineered FGF variants have revitalized the research interest into the therapeutic potential of certain additional FGFs (e.g. FGF1 and FGF15/FGF19). This review presents a brief overview of the FGF family, describing the mode of action of the different FGFs subgroups, and focuses on FGF1 and FGF15/FGF19, which appear to also represent promising new targets for the treatment of obesity and type 2 diabetes.

Functional epigenetic approach identifies frequently methylated genes in Ewing sarcoma.

Using a candidate gene approach we recently identified frequent methylation of the RASSF2 gene associated with poor overall survival in Ewing sarcoma (ES). To identify effective biomarkers in ES on a genome-wide scale, we used a functionally proven epigenetic approach, in which gene expression was induced in ES cell lines by treatment with a demethylating agent followed by hybridization onto high density gene expression microarrays. After following a strict selection criterion, 34 genes were selected for expression and methylation analysis in ES cell lines and primary ES. Eight genes (CTHRC1, DNAJA4, ECHDC2, NEFH, NPTX2, PHF11, RARRES2, TSGA14) showed methylation frequencies of>20% in ES tumors (range 24-71%), these genes were expressed in human bone marrow derived mesenchymal stem cells (hBMSC) and hypermethylation was associated with transcriptional silencing. Methylation of NPTX2 or PHF11 was associated with poorer prognosis in ES. In addition, six of the above genes also showed methylation frequency of>20% (range 36-50%) in osteosarcomas. Identification of these genes may provide insights into bone cancer tumorigenesis and development of epigenetic biomarkers for prognosis and detection of these rare tumor types.

RASSF2 methylation is a strong prognostic marker in younger age patients with Ewing sarcoma.

Ras-association domain family of genes consist of 10 members (RASSF1-RASSF10), all containing a Ras-association (RA) domain in either the C- or the N-terminus. Several members of this gene family are frequently methylated in common sporadic cancers; however, the role of the RASSF gene family in rare types of cancers, such as bone cancer, has remained largely uninvestigated. In this report, we investigated the methylation status of RASSF1A and RASSF2 in Ewing sarcoma (ES). Quantitative real-time methylation analysis (MethyLight) demonstrated that both genes were frequently methylated in Ewing sarcoma tumors (52.5% and 42.5%, respectively) as well as in ES cell lines and gene expression was upregulated in methylated cell lines after treatment with 5-aza-2'-deoxcytidine. Overexpression of either RASSF1A or RASSF2 reduced colony formation ability of ES cells. RASSF2 methylation correlated with poor overall survival (p = 0.028) and this association was more pronounced in patients under the age of 18 y (p = 0.002). These results suggest that both RASSF1A and RASSF2 are novel epigenetically inactivated tumor suppressor genes in Ewing sarcoma and RASSF2 methylation may have prognostic implications for ES patients.

Vacuolar-type H+-ATPase V1A subunit is a molecular partner of Wolfram syndrome 1 (WFS1) protein, which regulates its expression and stability.

Wolfram syndrome is an autosomal recessive disorder characterized by neurodegeneration and diabetes mellitus. The gene responsible for the syndrome (WFS1) encodes an endoplasmic reticulum (ER)-resident transmembrane protein that also localizes to secretory granules in pancreatic beta cells. Although its precise functions are unknown, WFS1 protein deficiency affects the unfolded protein response, intracellular ion homeostasis, cell cycle progression and granular acidification. In this study, immunofluorescent and electron-microscopy analyses confirmed that WFS1 also localizes to secretory granules in human neuroblastoma cells. We demonstrated a novel interaction between WFS1 and the V1A subunit of the H(+) V-ATPase (proton pump) by co-immunoprecipitation in human embryonic kidney (HEK) 293 cells and with endogenous proteins in human neuroblastoma cells. We mapped the interaction to the WFS1-N terminal, but not the C-terminal domain. V1A subunit expression was reduced in WFS1 stably and transiently depleted human neuroblastoma cells and depleted NT2 (human neuron-committed teratocarcinoma) cells. This reduced expression was not restored by adenoviral overexpression of BiP (immunoglobulin-binding protein) to correct the ER stress. Protein stability assays demonstrated that the V1A subunit was degraded more rapidly in WFS1 depleted neuroblastoma cells compared with wild-type; however, proteosomal inhibition did not restore the expression of the V1A subunit. Cell cycle assays measuring p21(cip) showed reduced levels in WFS1 depleted cells, and an inverse association between p21(cip) expression and apoptosis. We conclude that WFS1 has a specific interaction with the V1A subunit of H(+) ATPase; this interaction may be important both for pump assembly in the ER and for granular acidification.