Increased LRG1 Levels in Overweight and Obese Adolescents and Its Association with Obesity Markers, Including Leptin, Chemerin, and High Sensitivity C-Reactive Protein.

Leucine-rich α-2 glycoprotein1 (LRG1) is a member of the leucine-rich repeat (LRR) family that is implicated in multiple diseases, including cancer, aging, and heart failure, as well as diabetes and obesity. LRG1 plays a key role in diet-induced hepatosteatosis and insulin resistance by mediating the crosstalk between adipocytes and hepatocytes. LRG1 also promotes hepatosteatosis by upregulating de novo lipogenesis in the liver and suppressing fatty acid ß-oxidation. In this study, we investigated the association of LRG1 with obesity markers, including leptin and other adipokines in adolescents (11−14 years; n = 425). BMI-for-age classification based on WHO growth charts was used to define obesity. Plasma LRG1 was measured by ELISA, while other markers were measured by multiplexing assay. Median (IQR) of LRG1 levels was higher in obese (30 (25, 38) µg/mL) and overweight (30 (24, 39) µg/mL) adolescents, compared to normal-weight participants (27 (22, 35) µg/mL). The highest tertile of LRG1 had an OR [95% CI] of 2.55 [1.44, 4.53] for obesity. LRG1 was positively correlated to plasma levels of high sensitivity c-reactive protein (HsCRP) (ρ = 0.2), leptin (ρ = 0.2), and chemerin (ρ = 0.24) with p < 0.001. Additionally, it was positively associated with plasma level of IL6 (ρ = 0.17) and IL10 (ρ = 0.14) but not TNF-α. In conclusion, LRG1 levels are increased in obese adolescents and are associated with increased levels of adipogenic markers. These results suggest the usefulness of LRG1 as an early biomarker for obesity and its related pathologies in adolescents.

Overcoming Resistance to Endocrine Therapy in Breast Cancer: New Approaches to a Nagging Problem.

In the majority of women, breast cancer progresses through increased transcriptional activity due to over-expressed oestrogen receptors (ER). Therapeutic strategies include: (i) reduction of circulating ovarian oestrogens or of peripherally produced oestrogen (in postmenopausal women) with aromatase inhibitors and (ii) application of selective ER modulators for receptor blockade. The success of these interventions is limited by the variable but persistent onset of acquired resistance and by an intrinsic refractiveness which manifests despite adequate levels of ER in about 50% of patients with advanced metastatic disease. Loss of functional ER leads to endocrine insensitivity, loss of cellular adhesion and polarity, and increased migratory potential due to trans-differentiation of the epithelial cancer cells into a mesenchymal-like phenotype (epithelial-mesenchymal transition; EMT). Multiple mechanisms contributing to therapeutic failure have been proposed: (i) loss or modification of ER expression including epigenetic mechanisms, (ii) agonistic actions of selective ER modulators that may be enhanced through an increased expression of co-activators, (iii) attenuation of the tamoxifen metabolism through expression of genetic variants of P450 cytochromes which leads to more or less active metabolites and (iv) increased growth factor signalling particularly through epidermal growth factor receptor activation of pathways involving keratinocyte growth factor, platelet-derived growth factor, and nuclear factor x03BA;B. In addition, the small non-coding microRNAs, recently recognized as critical gene regulators, exhibit differential expression in tamoxifen-sensitive versus resistant cell lines. Several studies suggest the potential of using these either as targets or as therapeutic agents to modulate EMT regulators as a means of reversing the aggressive metastatic phenotype by reversal of the EMT, with the added benefit of re-sensitization to anti-oestrogens.

The Proinflammatory Role of ANGPTL8 R59W Variant in Modulating Inflammation through NF-κB Signaling Pathway under TNFα Stimulation.

BACKGROUND: Angiopoietin-like protein 8 (ANGPTL8) is known to regulate lipid metabolism and inflammation. It interacts with ANGPTL3 and ANGPTL4 to regulate lipoprotein lipase (LPL) activity and with IKK to modulate NF-κB activity. Further, a single nucleotide polymorphism (SNP) leading to the ANGPTL8 R59W variant associates with reduced low-density lipoprotein/high-density lipoprotein (LDL/HDL) and increased fasting blood glucose (FBG) in Hispanic and Arab individuals, respectively. In this study, we investigate the impact of the R59W variant on the inflammatory activity of ANGPTL8. METHODS: The ANGPTL8 R59W variant was genotyped in a discovery cohort of 867 Arab individuals from Kuwait. Plasma levels of ANGPTL8 and inflammatory markers were measured and tested for associations with the genotype; the associations were tested for replication in an independent cohort of 278 Arab individuals. Impact of the ANGPTL8 R59W variant on NF-κB activity was examined using approaches including overexpression, luciferase assay, and structural modeling of binding dynamics. RESULTS: The ANGPTL8 R59W variant was associated with increased circulatory levels of tumor necrosis factor alpha (TNFα) and interleukin 7 (IL7). Our in vitro studies using HepG2 cells revealed an increased phosphorylation of key inflammatory proteins of the NF-κB pathway in individuals with the R59W variant as compared to those with the wild type, and TNFα stimulation further elevated it. This finding was substantiated by increased luciferase activity of NF-κB p65 with the R59W variant. Modeled structural and binding variation due to R59W change in ANGPTL8 agreed with the observed increase in NF-κB activity. CONCLUSION: ANGPTL8 R59W is associated with increased circulatory TNFα, IL7, and NF-κB p65 activity. Weak transient binding of the ANGPTL8 R59W variant explains its regulatory role on the NF-κB pathway and inflammation.

Adolescent obesity and ANGPTL8: correlations with high sensitivity C-reactive protein, leptin, and chemerin.

Angiopoietin-like proteins (ANGPTLs) mediate many metabolic functions. We had recently reported increased plasma levels of ANGPTL8 in obese adults of Arab ethnicity. However, data on ANGPTL8 levels in adolescent obesity is lacking. Arab population is characterized by a rapid transition, due to sudden wealth seen in the post-oil era, in lifestyle, food habits and extent of physical activity. We adopted a cross-sectional study on Arab adolescents from Kuwait to examine the role of ANGPTL8 in adolescent obesity. The study cohort included 452 adolescents, aged 11-14 years, recruited from Middle Schools across Kuwait. BMI-for-age growth charts were used to categorize adolescents as normal-weight, overweight, and obese. ELISA and bead-based multiplexing assays were used to measure plasma levels of ANGPTL8 and other inflammation and obesity-related biomarkers. Data analysis showed significant differences in the plasma levels of ANGPTL8 among the three subgroups, with a significant increase in overweight and obese children compared to normal-weight children. This observation persisted even when the analysis was stratified by sex. Multinomial logistic regression analysis illustrated that adolescents with higher levels of ANGPTL8 were 7 times more likely to become obese and twice as likely to be overweight. ANGPTL8 levels were correlated with those of hsCRP, leptin and chemerin. ANGPTL8 level had a reasonable prognostic power for obesity with an AUC of 0.703 (95%-CI=0.648-0.759). These observations relating to increased ANGPTL8 levels corresponding to increased BMI-for-age z-scores indicate that ANGPTL8, along with hsCRP, leptin and chemerin, could play a role in the early stages of obesity development in children. ANGPTL8 is a potential early marker for adolescent obesity and is associated with well-known obesity and inflammatory markers.

Structural analysis of setmelanotide binding to MC4R variants in comparison to wild-type receptor.

AIMS: Melanocortin 4 receptor (MC4R) has a well-established role in regulating appetite, food intake and energy homeostasis. Setmelanotide is an MC4R agonist currently approved for weight loss in obese adults and children with mutations in components of the leptin-melanocortin pathway. This study aims to compare structural and functional aspects of the physiological MC4R agonist α-melanocyte-stimulating hormone (α-MSH) with setmelanotide. We also aim to show the binding affinity of setmelanotide to known MC4R human missense mutations associated with obesity. MAIN METHODS: AutoDock Vina was used in the structural analysis to calculate induced fit docking scores of ligand binding to MC4R wild type or the selected variants. HEK293-MC4R were utilized in the functional analysis of MC4R-actiavted pathways upon stimulating with α-MSH or setmelanotide. KEY FINDINGS: Our data shows that setmelanotide has a higher potency for cAMP formation and a weaker effect on ERK1/2 phosphorylation when compared to α-MSH indicating functional selectivity otherwise known as biased agonism. We also present structural data showing that setmelanotide has a higher binding affinity to MC4R compared to α-MSH. Lastly, we show that two loss-of-function and two gain-of-function MC4R variants change the conformation not only of the ligand binding pocket of the receptor but also of the peptide when bound to the receptor because the interaction network and the residues involved in the binding are altered. SIGNIFICANCE: Taken together, our study provides important insights into the diversity of MC4R signaling pathways which will facilitate the development of personalized anti-obesity drugs via refining MC4R agonists.

Urocortin Neuropeptide Levels Are Impaired in the PBMCs of Overweight Children.

The corticotropin-releasing hormone (CRH) and urocortins (UCNs) have been implicated in energy homeostasis and the cellular stress response. However, the expression of these neuropeptides in children remains unclear. Therefore, we determined the impact of obesity on their expression in 40 children who were normal weight, overweight, and had obesity. Peripheral blood mononuclear cells (PBMCs) and plasma were used to assess the expression of neuropeptides. THP1 cells were treated with 25 mM glucose and 200 µM palmitate, and gene expression was measured by real-time polymerase chain reaction (RT-PCR). Transcript levels of neuropeptides were decreased in PBMCs from children with increased body mass index as indicated by a significant decrease in UCN1, UCN3, and CRH mRNA in overweight and obese children. UCN3 mRNA expression was strongly correlated with UCN1, UCN2, and CRH. Exposure of THP1 cells to palmitate or a combination of high glucose and palmitate for 24 h increased CRH, UCN2, and UCN3 mRNA expression with concomitant increased levels of inflammatory and endoplasmic reticulum stress markers, suggesting a crosstalk between these neuropeptides and the cellular stress response. The differential impairment of the transcript levels of CRH and UCNs in PBMCs from overweight and obese children highlights their involvement in obesity-related metabolic and cellular stress.

Correlation of circulating ANGPTL5 levels with obesity, high sensitivity C-reactive protein and oxidized low-density lipoprotein in adolescents.

Angiopoietin-like proteins (ANGPTL) is a family of eight members known to play an important role in metabolic diseases. Of these, ANGPTL5 is suggested to regulate triglyceride metabolism and is increased in obesity and diabetes. However, its role in metabolic diseases in adolescents is not well-studied. In this study, we tested the hypothesis of a positive association between plasma ANGPTL5, and obesity, high sensitivity C-reactive protein (HsCRP) and oxidized low-density lipoprotein (Ox-LDL) in adolescents. Adolescents (N = 431; age 11-14 years) were randomly selected from middle schools in Kuwait. Obesity was classified by the BMI-for-age based on the WHO growth charts. Plasma ANGPTL5, HsCRP, and Ox-LDL were measured using ELISA. The prevalence of overweight and obesity was 20.65% and 33.18%, respectively. Mean (SD) plasma ANGPTL5 levels were significantly higher in obese, compared with overweight and normal-weight adolescents (23.05 (8.79) vs 18.39 (7.08) ng/mL, and 18.26 (6.95) ng/ml, respectively). ANGPTL5 was positively associated with both HsCRP (ρ=0.27, p < 0.001) and Ox-LDL (ρ = 0.24, p < 0.001). In Conclusion, ANGPTL5 levels are elevated in obese adolescents and are associated with cardiovascular disease risk factors, HsCRP and Ox-LDL. The use of ANGPTL5 as a powerful diagnostic and prognostic tool in obesity and metabolic diseases needs to be further evaluated.

Hypomagnesemia in diabetes patients: comparison of serum and intracellular measurement of responses to magnesium supplementation and its role in inflammation.

PURPOSE: In this clinical trial, we assessed the efficacy of magnesium (Mg) supplementation in hypomagnesemic type 2 diabetes patients in restoring serum and intracellular Mg levels. The study had two coprimary end points: the change in serum and intracellular Mg level between baseline and after 3 months of supplementation. We compared the efficacy with regard to lowering hemoglobin A1c (HbA1c), C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), and 8-isoprostane as secondary end points. PATIENTS AND METHODS: In an open-label trial, 47 hypomagnesemic type 2 diabetes patients were administered 336 mg Mg daily. At baseline and after 3 months, serum, cellular Mg, and inflammation biomarkers were measured. For intracellular Mg levels, sublingual epithelial cells were analyzed by analytical scanning electron microscopy using computerized elemental X-ray analysis. Blood samples were analyzed for Mg, creatinine, HbA1c, and CRP. Systemic inflammatory markers including TNF-α and the oxidative stress marker 8-isoprostane were determined using enzyme-linked immunosorbent assay. RESULTS: Mg supplementation significantly increased the intracellular and serum levels. Statistically clinical improvement in HbA1c and CRP levels was not observed, but significant decreases in TNF-α as well as in 8-isoprostane were found. CONCLUSION: A feasible clinical method for the assessment of intracellular Mg was demonstrated in tissue samples obtained noninvasively, providing evidence for potential clinical translation of this method to routinely determine intracellular Mg concentration.

Bleb formation is induced by alkaline but not acidic pH in estrogen receptor silenced breast cancer cells.

De novo and acquired resistance to endocrine-based therapies in breast cancer occurs in parallel with epithelial to mesenchymal transition (EMT), which is associated with enhanced proliferative and metastatic potential, and poor clinical outcome. We have established several endocrine insensitive breast cancer lines by shRNA-induced depletion of estrogen receptor (ER) by transfection of MCF7 cells. All of these exhibit EMT. We have previously reported that brief exposure of specifically ER- breast cancer cells, to extracellular alkaline pH, results in cell rounding and segregation, and leads to enhanced invasive potential. In this study we describe more detailed morphological changes and compare these with cell exposure to acidic pH. Morphological changes and localization of various molecules critical for cell adhesion and motility, associated with pH effects, were assessed by live cell microscopy, electron microscopy, and immunofluorescence. Exposure of either ER- or ER+ breast cancer cells to extracellular acidic pH did not induce significant changes in morphological appearance. Conversely, brief exposure of specifically ER silenced cells, to alkaline pH, resulted in cell contractolation and formation of bleb-like actin-rich structures which were evenly distributed on the outer membrane. Integrin α2, FAK, and JAM-1 were found in the cytoplasm streaming into the newly formed blebs. These blebs appear to be related to cell polarity and movement. Pre-treatment with cytochalasin-D or inhibitors of Rho or MLCK prevented both contractolation and bleb formation. Our data suggest that the effect of pH on the microenvironment of endocrine resistant breast cancer cells needs to be more extensively investigated. Alkaline, rather than acidic pH, appears to induce dramatic morphological changes, and enhances their invasive capabilities, through re-organization of cortical actin.