Henk S. Schipper

Natural killer T cells in adipose tissue prevent insulin resistance

Lipid overload and adipocyte dysfunction are key to the development of insulin resistance and can be induced by a high-fat diet. CD1d-restricted invariant natural killer T (iNKT) cells have been proposed as mediators between lipid overload and insulin resistance, but recent studies found decreased iNKT cell numbers and marginal effects of iNKT cell depletion on insulin resistance under high-fat diet conditions. Here, we focused on the role of iNKT cells under normal conditions. We showed that iNKT cell-deficient mice on a low-fat diet, considered a normal diet for mice, displayed a distinctive insulin resistance phenotype without overt adipose tissue inflammation. Insulin resistance was characterized by adipocyte dysfunction, including adipocyte hypertrophy, increased leptin, and decreased adiponectin levels. The lack of liver abnormalities in CD1d-null mice together with the enrichment of CD1d-restricted iNKT cells in both mouse and human adipose tissue indicated a specific role for adipose tissue-resident iNKT cells in the development of insulin resistance. Strikingly, iNKT cell function was directly modulated by adipocytes, which acted as lipid antigen-presenting cells in a CD1d-mediated fashion. Based on these findings, we propose that, especially under low-fat diet conditions, adipose tissue-resident iNKT cells maintain healthy adipose tissue through direct interplay with adipocytes and prevent insulin resistance.

A Multiplex Immunoassay for Human Adipokine Profiling

BACKGROUND Adipose tissue secretory proteins, called adipokines, play pivotal roles in the pathophysiology of obesity and its associated disorders such as metabolic syndrome, type 2 diabetes, and cardiovascular disease. Because methods for comprehensive adipokine profiling in patient plasma and other biological samples are currently limited, we developed a multiplex immunoassay for rapid and high-throughput measurement of 25 adipokines in only 50 microL of sample. METHODS (Pre)adipocyte and ex vivo cultured adipose tissue supernatants were generated and together with plasma from 5 morbidly obese patients and 5 healthy and normal weight controls used to develop the adipokine multiplex immunoassay and test its usefulness in biological samples. We assessed adipokine dynamic ranges, lower limits of detection and quantification, cross-reactivity, intra- and interassay variation, and correlation with adipokine ELISAs. RESULTS The limits of quantification and broad dynamic ranges enabled measurement of all 25 adipokines in supernatants and patient plasmas, with the exception of TNF-alpha in plasma samples. Intraassay variation was <10% for all adipokines; interassay variation was < 15%. The multiplex immunoassay results correlated significantly with ELISA measurements. Plasma adipokine profiling showed significantly higher concentrations of the novel adipokines cathepsin S (5.1 x 10(4) vs 4.3 x 10(4) ng/L, P = 0.003) and chemerin (4.1 x 10(5) vs 2.7 x 10(5) ng/L, P = 0.0008) in morbidly obese patients than normal weight controls, besides the established differences in adiponectin and leptin concentrations. CONCLUSIONS Our findings underscore the relevance of the novel adipokines cathepsin S and chemerin, but foremost the potential of this novel method for both comprehensive adipokine profiling in large patient cohorts and for biological discovery.

Vitamin D deficiency in childhood obesity is associated with high levels of circulating inflammatory mediators, and low insulin sensitivity

Hypothesis:Childhood obesity is accompanied by low-grade systemic inflammation, which contributes to the development of insulin resistance and cardiovascular complications later in life. As vitamin D exhibits profound immunomodulatory functions and vitamin D deficiency is highly prevalent in childhood obesity, we hypothesized that vitamin D deficiency in childhood obesity coincides with enhanced systemic inflammation and reduced insulin sensitivity.Methods:In a cross-sectional study of 64 obese and 32 healthy children aged 6–16 years, comprehensive profiling of 32 circulating inflammatory mediators was performed, together with assessment of 25-hydroxyvitamin D (25(OH)D) levels and measures for insulin sensitivity.Results:Severe vitamin D insufficiency, which is further referred to as vitamin D deficiency, was defined as a 25(OH)D level ⩽37.5 nmol l–1, and was highly prevalent in obese (56%) versus healthy control children (16%). Throughout the study, 25(OH)D-deficient children were compared with the other children, including 25(OH)D insufficient (37.5–50 nmol l–1) and 25(OH)D sufficient children (⩾50 nmol l–1). First, 25(OH)D-deficient obese children showed a lower insulin sensitivity than other obese children, as measured by a lower quantitative insulin sensitivity check index. Second, the association between 25(OH)D deficiency and insulin resistance in childhood obesity was confirmed with multiple regression analysis. Third, 25(OH)D-deficient obese children showed higher levels of the inflammatory mediators cathepsin S, chemerin and soluble vascular adhesion molecule (sVCAM), compared with the other obese children. Finally, hierarchical cluster analysis revealed an over-representation of 25(OH)D deficiency in obese children expressing inflammatory mediator clusters with high levels of cathepsin S, sVCAM and chemerin.Conclusion:25(OH)D deficiency in childhood obesity was associated with enhanced systemic inflammation and reduced insulin sensitivity. The high cathepsin S and sVCAM levels may reflect activation of a pro-inflammatory, pro-diabetic and atherogenic pathway, which could be inhibited by vitamin D supplementation.

Human adipocyte extracellular vesicles in reciprocal signaling between adipocytes and macrophages

Extracellular vesicles (EVs) released by human adipocytes or adipose tissue (AT)‐explants play a role in the paracrine interaction between adipocytes and macrophages, a key mechanism in AT inflammation, leading to metabolic complications like insulin resistance (IR) were determined.

Altered Plasma Adipokine Levels and in Vitro Adipocyte Differentiation in Pediatric Type 1 Diabetes

CONTEXT Type 1 diabetes (T1D) is considered a proinflammatory condition. Adipose tissue involvement seems evident because adiponectin levels correlate with disease remission and administration of leptin suppresses the low-grade systemic inflammation in mice with T1D. Whether adipose tissue involvement in T1D already occurs at a young age is yet unknown. OBJECTIVE The aim was to explore the extent of adipokine alterations in pediatric T1D and gain more insight into the mechanisms underlying the involvement of adipose tissue. DESIGN AND PARTICIPANTS First, plasma adipokine profiling (24 adipokines) of 20 children with onset T1D, 20 children with long-standing T1D, and 17 healthy controls was performed using a recently developed and validated multiplex immunoassay. Second, the effects of diabetic plasma factors on preadipocyte proliferation and differentiation were studied in vitro. RESULTS In children with onset and long-standing T1D, plasma adipokine profiling showed increased levels of various adipokines acting at the crossroads of adipose tissue function and inflammation, including CCL2/monocyte chemoattractant protein-1 and the novel adipokines cathepsin S, chemerin, and tissue inhibitor of metalloproteinase-1 (P < 0.05). Furthermore, onset and long-standing diabetic plasma significantly induced preadipocyte proliferation and adipocyte differentiation in vitro (P < 0.05). Two candidate plasma factors, glucose and the saturated fatty acid palmitic acid, did not affect proliferation or adipocyte differentiation in vitro but were found to increase CCL2 (monocyte chemoattractant protein-1) secretion by adipocytes. CONCLUSIONS The adipogenic effects of diabetic plasma in vitro and the altered adipokine levels in vivo suggest adipose tissue involvement in the low-grade inflammation associated with T1D, already in pediatric patients.

Differential adipokine receptor expression on circulating leukocyte subsets in lean and obese children

Background Childhood obesity prevalence has increased worldwide and is an important risk factor for type 2 diabetes (T2D) and cardiovascular disease (CVD). The production of inflammatory adipokines by obese adipose tissue contributes to the development of T2D and CVD. While levels of circulating adipokines such as adiponectin and leptin have been established in obese children and adults, the expression of adiponectin and leptin receptors on circulating immune cells can modulate adipokine signalling, but has not been studied so far. Here, we aim to establish the expression of adiponectin and leptin receptors on circulating immune cells in obese children pre and post-lifestyle intervention compared to normal weight control children. Methods 13 obese children before and after a 1-year lifestyle intervention were compared with an age and sex-matched normal weight control group of 15 children. Next to routine clinical and biochemical parameters, circulating adipokines were measured, and flow cytometric analysis of adiponectin receptor 1 and 2 (AdipoR1, AdipoR2) and leptin receptor expression on peripheral blood mononuclear cell subsets was performed. Results Obese children exhibited typical clinical and biochemical characteristics compared to controls, including a higher BMI-SD, blood pressure and circulating leptin levels, combined with a lower insulin sensitivity index (QUICKI). The 1-year lifestyle intervention resulted in stabilization of their BMI-SD. Overall, circulating leukocyte subsets showed distinct adipokine receptor expression profiles. While monocytes expressed high levels of all adipokine receptors, NK and iNKT cells predominantly expressed AdipoR2, and B-lymphocytes and CD4+ and CD8+ T-lymphocyte subsets expressed AdipoR2 as well as leptin receptor. Strikingly though, leukocyte subset numbers and adipokine receptor expression profiles were largely similar in obese children and controls. Obese children showed higher naïve B-cell numbers, and pre-intervention also higher numbers of immature transition B-cells and intermediate CD14++CD16+ monocytes combined with lower total monocyte numbers, compared to controls. Furthermore, adiponectin receptor 1 expression on nonclassical CD14+CD16++ monocytes was consistently upregulated in obese children pre-intervention, compared to controls. However, none of the differences in leukocyte subset numbers and adipokine receptor expression profiles between obese children and controls remained significant after multiple testing correction. Conclusions First, the distinct adipokine receptor profiles of circulating leukocyte subsets may partly explain the differential impact of adipokines on leukocyte subsets. Second, the similarities in adipokine receptor expression profiles between obese children and normal weight controls suggest that adipokine signaling in childhood obesity is primarily modulated by circulating adipokine levels, instead of adipokine receptor expression.

PS15 - 73. Identification and characterization of microvesicles secreted by human SGBS-adipocytes

Obesity and its metabolic complications are important risk factors for cardiovascular disease. Obesity is associated with adipose tissue (AT) inflammation, initiated by adipocyte hypertrophy and ultimately resulting in low-grade systemic inflammation. Complex intercellular communication between adipocytes and AT-resident immune cells underlie AT inflammation.

PS15 - 74. CD1d-restricted NKT cell function prevents insulin resistance in lean mice, and is regulated by adipocytes

Lipid overload and adipocyte dysfunction are key to the development of insulin resistance and can be induced by a high fat diet (HFD). CD1d-restricted iNKT cells have been proposed as mediators between lipid overload and insulin resistance, but recent studies found decreased iNKT cell numbers and marginal effects of iNKT cell depletion on insulin resistance under HFD conditions.

Abstract 705: Interfering with mesenchymal stem cell-induced chemoresistance via COX-1 and thromboxane synthase inhibition

Background: Environment-mediated resistance to chemotherapy is emerging as a cause of treatment failure. We showed that mesenchymal stem cells (MSCs) induce resistance to a broad spectrum of chemotherapies. Upon platinum stimulation, two unique polyunsaturated fatty acids (PIFAs), 12-oxo-5,8,10-heptadecatrienoic acid (KHT(n-6)) and hexadeca-4,7,10,13-tetraenoic acid (16:4(n-3)) are produced by MSCs, which, via a paracrine mechanism, induce chemoresistance in tumors. Aim: Here, we aim to prevent MSC-induced chemoresistance by blocking PIFA production. Method: Various mouse models were established in which recruitment of MSCs to tumors was mimicked by iv administration of MSCs. Furthermore, cultured MSCs were treated with various drugs in order to block their chemo-protective capacity. Conditioned media were subsequently analyzed in vivo. Results: Conditioned medium of platinum-stimulated, cultured MSCs induced chemoresistance in tumor-bearing mice. However, the capacity to secrete the chemo-protective PIFAs was only retained by mesenchymal cells with multi-lineage differentiation potential: MSCs and mouse embryonic fibroblasts (MEFs). More differentiated progeny from the mesenchymal lineage including 3T3 Fibroblasts, pre-adipocytes (3T3-L1), differentiated adipocytes, pre-osteoblasts (MC3T3) and differentiated osteoblasts were not capable of PIFA production upon platinum stimulation. Furthermore, this cytoprotective response was specific for multipotent cells from the mesenchymal lineage, since administration of hematopoietic stem cells did not influence the tumor response to chemotherapy. One of the identified PIFAs, KHT(n-6), is known to be a by-product of thromboxane A2 synthesis, which is a downstream product of the cyclooxygenase (COX)-1 and thromboxane synthase (TxS) pathway. Interestingly, conditioned media from MSCs pre-treated with SC-560, a highly selective COX-1 inhibitor, indomethacin, a relatively selective COX-1 inhibitor or one of two specific TxS inhibitors (ozagrel and furegrelate) did not induce chemoresistance in vivo. Of note, selective COX-2 inhibition in MSC by celecoxib did not affect their capacity to induce chemoresistance, suggestive of a specific COX-1/TxS-dependent PIFA production. When mice were treated with either indomethacin or ozagrel as single agents no anti-tumor effect was observed. However, combining these drugs with cisplatin in vivo had an additive anti-tumor effect compared to cisplatin alone. Conclusion: We show a novel mechanism of chemoresistance mediated by PIFA released from early, multipotent cells of the mesenchymal lineage. The production of these PIFAs is dependent on COX-1 and TxS, and blocking these enzymes enhanced the antitumor effects of cisplatin in vivo, making these enzymes drugable targets to prevent PIFA-induced chemoresistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 705. doi:10.1158/1538-7445.AM2011-705