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Summary: Background: Eighty percent of adolescents with severe obesity suffer from non‐alcoholic fatty liver disease (NAFLD). Non‐invasive prediction models have been tested in adults, however, they performed poorly in paediatric populations. Objective: This study aimed to investigate novel biomarkers for NAFLD and to develop a score that predicts liver fat in youth with severe obesity. Methods: From a population with a BMI >97th percentile aged 9‐19 years (n = 68), clinically thoroughly characterized including MRI‐derived proton density fat fraction (MRI‐PDFF), amino acids and acylcarnitines were measured by HPLC‐MS. Results: In children with NAFLD, higher levels of plasma branched‐chain amino acids (BCAA) were determined. BCAAs correlated with MRI‐PDFF (R = 0.46, p <.01). We identified a linear regression model adjusted for age, sex and pubertal stage consisting of BCAAs, ALT, GGT, ferritin and insulin that predicted MRI‐PDFF (R = 0.75, p <.01). ROC analysis of this model revealed AUCs of 0.85, 0.85 and 0.92 for the detection of any, moderate and severe steatosis, respectively, thus markedly outperforming previously published scores. Conclusion: BCAAs could be an important link between obesity and other metabolic pathways. A BCAA‐based metabolic score can predict steatosis grade in high‐risk children and adolescents and may provide a feasible alternative to sophisticated methods like MRI or biopsy in the future. [ABSTRACT FROM AUTHOR]

Background & aims: Osteopontin, a multifunctional protein and inflammatory cytokine, is overexpressed in adipose tissue and liver in obesity and contributes to the induction of adipose tissue inflammation and non‐alcoholic fatty liver (NAFL). Studies performed in both mice and humans also point to a potential role for OPN in malignant transformation and tumour growth. To fully understand the role of OPN on the development of NAFL‐derived hepatocellular carcinoma (HCC), we applied a non‐alcoholic steatohepatitis (NASH)‐HCC mouse model on osteopontin‐deficient (Spp1−/−) mice analysing time points of NASH, fibrosis and HCC compared to wild‐type mice. Methods: Two‐day‐old wild‐type and Spp1−/− mice received a low‐dose streptozotocin injection in order to induce diabetes, and were fed a high‐fat diet starting from week 4. Different cohorts of mice of both genotypes were sacrificed at 8, 12 and 19 weeks of age to evaluate the NASH, fibrosis and HCC phenotypes respectively. Results: Spp1−/− animals showed enhanced hepatic lipid accumulation and aggravated NASH, as also increased hepatocellular apoptosis and accelerated fibrosis. The worse steatotic and fibrotic phenotypes observed in Spp1−/− mice might be driven by enhanced hepatic fatty acid influx through CD36 overexpression and by a pathological accumulation of specific diacylglycerol species during NAFL. Lack of osteopontin lowered systemic inflammation, prevented HCC progression to less differentiated tumours and improved overall survival. Conclusions: Lack of osteopontin dissociates NASH‐fibrosis severity from overall survival and HCC malignant transformation in NAFLD, and is therefore a putative therapeutic target only for advanced chronic liver disease. [ABSTRACT FROM AUTHOR]

Background and Aim Obesity is a major risk factor for liver fibrosis and tightly associated with low levels of adiponectin. Adiponectin has antifibrogenic activity protecting from liver fibrosis, which is mainly driven by activated hepatic stellate cells (HSC). Aquaporins are transmembrane proteins that allow the movement of water and, in case of aquaglyceroporins (AQPs), of glycerol that is needed in quiescent HSC for lipogenesis. Expression of various AQPs in liver is altered by obesity; however, the mechanisms through which obesity influences HSCs activation and AQPs expression remain unclear. This study aimed to identify obesity-associated factors that are related to HSC AQPs expression activation and lipid storage. Methods Correlations between serum adipokine levels and hepatic AQPs gene expression were analyzed from a cohort of obese patients. AQP and fibrotic gene expression was determined in a HSC line (LX2) and in a hepatocyte cell line (HepG2) after stimulation with adiponectin using quantitative real-time polymerase chain reaction. Results We found that serum adiponectin significantly correlated with liver AQP3, AQP7, AQP9 gene expressions. In vitro, adiponectin induced upregulation of AQP3 gene and AQP3 protein expression in human HSCs, but not in hepatocytes, while AQP7, AQP9 remained undetectable. Accordingly, HSC stimulated with adiponectin increased glycerol uptake, lipogenic gene expression, and lipid storage while downregulating activation/fibrosis markers. Conclusions These findings demonstrate that adiponectin is a potent inhibitor of HSC activation and induces AQPs expression. Thus, low serum levels of adiponectin could be a mechanism how obesity affects the functional state of HSC, thereby contributing to obesity-associated liver fibrosis. [ABSTRACT FROM AUTHOR]

Background Infiltration of white adipose tissue ( WAT) by inflammatory cells in obesity is considered to be a key event in the development of insulin resistance. Recently, mast cells ( MCs) have been identified as new players in the pathogenesis of obesity. We aimed to investigate the relationship between MCs and various inflammatory markers in serum and WAT and to determine the role of MCs in the aetiology of insulin resistance. Materials and methods Gene expression was measured in WAT from 20 morbidly obese patients and 20 nonobese control subjects. Homoeostasis Model of Assessment-Insulin Resistance ( HOMA- IR) was used to estimate insulin sensitivity. In addition, wild-type and mast cell-deficient mice were fed a high-fat or low-fat diet to study mast cell influence on inflammatory cell polarization in WAT and overall metabolic changes. Results WAT levels of MC-specific TPSb2 transcript were increased in obesity and significantly positively correlated with TNF, CCL2, CCL5 and CD68 gene expression levels in our study subjects after adjustment for sex, age and BMI. Accordingly, MC deficiency abrogated increase in expression of pro-inflammatory M1 macrophage marker genes in mouse WAT upon high-fat diet feeding. However, MCs accumulated in obese human WAT independent of insulin resistance and systemic changes in inflammatory mediators. Conclusions Our results suggest that MCs contribute to the local pro-inflammatory state within WAT in obesity but do not play a primary role in causing insulin resistance. [ABSTRACT FROM AUTHOR]

Objective: Macrophages are the main drivers of obesity-induced adipose tissue (AT) inflammation that causes insulin resistance. Macrophages polarize toward different inflammatory (M1) or protective (M2) phenotypes. Osteopontin (OPN) is an inflammatory cytokine highly expressed in AT in obesity and known to be involved in chronic inflammatory processes. It was hypothesized that OPN polarizes macrophages into a proinflammatory phenotype.Methods: AT macrophages (ATMs) of OPN-deficient (Spp1(-/-) ) and wild-type C57BL/6 (WT) mice with obesity and bone marrow-derived macrophages (BMDMs) of Spp1(-/-) and WT mice as well as human monocyte-derived macrophages (MDMs) polarized in the presence of OPN were investigated.Results: While ATM infiltration was lower in Spp1(-/-) upon high-fat diet, Spp1(-/-) ATMs expressed more M1 and less M2 markers but less tumor necrosis factor-α compared with WT. There was no effect of OPN deficiency on BMDM polarization. In human MDMs, the presence of OPN during polarization ambiguously altered M1/M2-related marker expression and diminished LPS-induced inflammatory cytokine production. Strikingly, phagocytic activity was elevated by the presence of OPN during polarization in both human MDMs and murine BMDMs.Conclusions: In contradiction to our hypothesis, data indicated that OPN does not induce inflammatory macrophages but was a signal to induce phagocytosis, which may be required due to increased adipocyte death in obesity. [ABSTRACT FROM AUTHOR]

Objective Osteopontin (OPN) is upregulated in adipose tissue (AT) in obesity and contributes to subclinical inflammation, adipocyte dysfunction, and insulin resistance. OPN effects can be increased by cleavage by matrix metalloproteinases (MMP). This study aimed at investigating the presence of OPN cleavage products in human AT in obesity and their impact on adipocyte function and immunological blockade of these effects. Methods AT of severely obese and control donors was investigated for OPN and MMP expression and the presence of OPN cleavage fragments. Primary adipocytes were isolated from human donors for in vitro investigation of cleaved OPN effects. Results OPN and MMP-9 expression was highly correlated in AT from obese donors, and increased levels of cleaved OPN were detected in AT from obese individuals. The in vitro effect of OPN on adipocyte inflammation and insulin resistance was enhanced by protease cleavage, which could finally be blocked with a monoclonal antibody directed against the MMP cleavage site of OPN. Conclusions These findings show that MMP cleavage of OPN in AT occurs in obesity, thereby enhancing OPN's inflammatory and pro-diabetic activity on adipocytes. Specifically targeting MMP-cleaved OPN opens avenues for prevention and treatment of obesity-induced type 2 diabetes. [ABSTRACT FROM AUTHOR]

Accumulation of cytotoxic and T-helper (Th)1 cells together with a loss of regulatory T cells in gonadal adipose tissue was recently shown to contribute to obesity-induced adipose tissue inflammation and insulin resistance in mice. Human data on T-cell populations in obese adipose tissue and their potential functional relevance are very limited. We aimed to investigate abundance and proportion of T-lymphocyte sub-populations in human adipose tissue in obesity and potential correlations with anthropometric data, insulin resistance, and systemic and adipose tissue inflammation. Therefore, we analyzed expression of marker genes specific for pan-T cells and T-cell subsets in visceral and subcutaneous adipose tissue from highly obese patients (BMI >40 kg/m2, n = 20) and lean to overweight control subjects matched for age and sex (BMI <30 kg/m2; n = 20). All T-cell markers were significantly upregulated in obese adipose tissue and correlated with adipose tissue inflammation. Proportions of cytotoxic T cells and Th1 cells were unchanged, whereas those of regulatory T cells and Th2 were increased in visceral adipose tissue from obese compared to control subjects. Systemic and adipose tissue inflammation positively correlated with the visceral adipose abundance of cytotoxic T cells and Th1 cells but also regulatory T cells within the obese group. Therefore, this study confirms a potential role of T cells in human obesity-driven inflammation but does not support a loss of protective regulatory T cells to contribute to adipose tissue inflammation in obese patients as suggested by recent animal studies. [ABSTRACT FROM AUTHOR]

In recent years the potential application of the immunomodulatory effects of polyunsaturated FA (PUFA), particularly those of the n−3 series, in a variety of inflammatory disorders has been of considerable interest. However, the mechanisms underlying inhibition of T-cell activation have so far been unclear. In this short review we summarize possible mechanisms for the modulation of immune responses by PUFA. Effects of PUFA on T-cell signal transduction pathways and underlying molecular mechanisms are described in detail. These recent results add considerably to the understanding of mechanisms of PUFA actions, but their relevance in the in vivo situation must still be elucidated. [ABSTRACT FROM AUTHOR]

Objective: Leflunomide, a potent disease-modifying antirheumatic drug of the isoxazole class, exhibits antiinflammatory, antiproliferative, and immunosuppressive effects by largely unknown mechanisms, although alterations of pyrimidine synthesis have been proposed. Successful immune responsiveness requires T cell activation by interaction with antigen-presenting cells (APCs), and integrin activation and formation of an immunologic synapse (IS). In this study, we evaluated the impact of the active leflunomide metabolite teriflunomide on T cell integrin activation, evolution of the IS, and antigen-specific formation of stable T cell/APC conjugates., Methods: Effects of pharmacologic concentrations of teriflunomide on CD3/CD28- and lymphocyte function-associated antigen 1-induced signal transduction and activation of primary human T cells were investigated. Furthermore, T cells were stimulated with superantigen- and antigen-pulsed APCs to study relocalization of molecules to the IS and T cell/APC conjugate formation., Results: Teriflunomide inhibited T cell receptor (TCR)/CD3-mediated calcium mobilization, but other critical T cell signaling events, including activation of MAPK and NF-kappaB, remained unaltered. In contrast, inhibition of TCR/CD3-triggered beta1,2 integrin avidity and integrin-mediated costimulation (outside-in signaling) by teriflunomide revealed a striking interference with integrin function that was independent of altered pyrimidine synthesis. Moreover, teriflunomide abolished molecule relocalization to the IS and induction of T cell/APC conjugates., Conclusion: These data show that the active metabolite of leflunomide prevents the interaction of T cells with APCs to form an IS. Since IS formation is crucial for eliciting an immune response, this novel mechanism could underlie the beneficial effects of leflunomide in immune-mediated disorders such as rheumatoid arthritis.