Jaap G. Neels

A Subpopulation of Macrophages Infiltrates Hypertrophic Adipose Tissue and Is Activated by Free Fatty Acids via Toll-like Receptors 2 and 4 and JNK-dependent Pathways

Obesity and type 2 diabetes are characterized by decreased insulin sensitivity, elevated concentrations of free fatty acids (FFAs), and increased macrophage infiltration in adipose tissue (AT). Here, we show that FFAs can cause activation of RAW264.7 cells primarily via the JNK signaling cascade and that TLR2 and TLR4 are upstream of JNK and help transduce FFA proinflammatory signals. We also demonstrate that F4/80+CD11b+CD11c+ bone marrow-derived dendritic cells (BMDCs) have heightened proinflammatory activity compared with F4/80+CD11b+CD11c- bone marrow-derived macrophages and that the proinflammatory activity and JNK phosphorylation of BMDCs, but not bone marrow-derived macrophages, was further increased by FFA treatment. F4/80+CD11b+CD11c+ cells were found in AT, and the proportion and number of these cells in AT is increased in ob/ob mice and by feeding wild type mice a high fat diet for 1 and 12 weeks. AT F4/80+CD11b+CD11c+ cells express increased inflammatory markers compared with F4/80+CD11b+CD11c- cells, and FFA treatment increased inflammatory responses in these cells. In addition, we found that CD11c expression is increased in skeletal muscle of high fat diet-fed mice and that conditioned medium from FFA-treated wild type BMDCs, but not TLR2/4 DKO BMDCs, can induce insulin resistance in L6 myotubes. Together our results show that FFAs can activate CD11c+ myeloid proinflammatory cells via TLR2/4 and JNK signaling pathways, thereby promoting inflammation and subsequent cellular insulin resistance.

The Light Chain of Factor VIII Comprises a Binding Site for Low Density Lipoprotein Receptor-related Protein

In the present study, the interaction between the endocytic receptor low density lipoprotein receptor-related protein (LRP) and coagulation factor VIII (FVIII) was investigated. Using purified components, FVIII was found to bind to LRP in a reversible and dose-dependent manner (K d ≈ 60 nm). The interaction appeared to be specific because the LRP antagonist receptor-associated protein readily inhibited binding of FVIII to LRP (IC50 ≈ 1 nm). In addition, a 12-fold molar excess of the physiological carrier of FVIII,i.e. von Willebrand factor (vWF), reduced the binding of FVIII to LRP by over 90%. Cellular degradation of125I-labeled FVIII by LRP-expressing cells (≈ 8 fmol/105 cells after a 4.5-h incubation) was reduced by approximately 70% in the presence of receptor-associated protein. LRP-directed antibodies inhibited degradation to a similar extent, indicating that LRP indeed contributes to binding and transport of FVIII to the intracellular degradation pathway. Degradation of FVIII was completely inhibited by vWF. Because vWF binding by FVIII involves its light chain, LRP binding to this subunit was studied. In ligand blotting experiments, binding of FVIII light chain to LRP could be visualized. More detailed analysis revealed that FVIII light chain interacts with LRP with moderate affinity (k on≈ 5 × 104 m −1s−1; k off ≈ 2.5 × 10−3 s−1; K d ≈ 50 nm). Furthermore, experiments using recombinant FVIII C2 domain showed that this domain contributes to the interaction with LRP. In contrast, no association of FVIII heavy chain to LRP could be detected under the same experimental conditions. Collectively, our data demonstrate that in vitro LRP is able to bind FVIII at the cell surface and to mediate its transport to the intracellular degradation pathway. FVIII-LRP interaction involves the FVIII light chain, and FVIII-vWF complex formation plays a regulatory role in LRP binding. Our findings may explain the beneficial effect of vWF on thein vivo survival of FVIII.

THE SECOND AND FOURTH CLUSTER OF CLASS A CYSTEINE-RICH REPEATS OF THE LOW DENSITY LIPOPROTEIN RECEPTOR-RELATED PROTEIN SHARE LIGAND-BINDING PROPERTIES

The low density lipoprotein receptor-related protein (LRP) is a multifunctional endocytic cell-surface receptor that binds and internalizes a diverse array of ligands. The receptor contains four putative ligand-binding domains, generally referred to as clusters I, II, III, and IV. In this study, soluble recombinant receptor fragments, representing each of the four individual clusters, were used to map the binding sites of a set of structurally and functionally distinct ligands. Using surface plasmon resonance, we studied the binding of these fragments to methylamine-activated α2-macroglobulin, pro-urokinase-type plasminogen activator, tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1, t-PA·plasminogen activator inhibitor-1 complexes, lipoprotein lipase, apolipoprotein E, tissue factor pathway inhibitor, lactoferrin, the light chain of blood coagulation factor VIII, and the intracellular chaperone receptor-associated protein (RAP). No binding of the cluster I fragment to any of the tested ligands was observed. The cluster III fragment only bound to the anti-LRP monoclonal antibody α2MRα3 and weakly to RAP. Except for t-PA, we found that each of the ligands tested binds both to cluster II and to cluster IV. The affinity rate constants of ligand binding to clusters II and IV and to LRP were measured, showing that clusters II and IV display only minor differences in ligand-binding kinetics. Furthermore, we demonstrate that the subdomains C3–C7 of cluster II are essential for binding of ligands and that this segment partially overlaps with a RAP-binding site on cluster II. Finally, we show that one RAP molecule can bind to different clusters simultaneously, supporting a model in which RAP binding to LRP induces a conformational change in the receptor that is incompatible with ligand binding.

Glucocorticoids and Thiazolidinediones Interfere with Adipocyte-mediated Macrophage Chemotaxis and Recruitment

The link between intra-abdominal adiposity and type II diabetes has been known for decades, and adipose tissue macrophage (ATM)-associated inflammation has recently been linked to insulin resistance. However, the mechanisms associated with ATM recruitment remain ill defined. Herein, we describe in vitro chemotaxis studies, in which adipocyte conditioned medium was used to stimulate macrophage migration. We demonstrate that tumor necrosis factor α and free fatty acids, key inflammatory stimuli involved in obesity-associated autocrine/paracrine inflammatory signaling, stimulate adipocyte expression and secretion of macrophage chemoattractants. Pharmacological studies showed that peroxisome proliferator-activated receptor γ agonists and glucocorticoids potently inhibit adipocyte- induced recruitment of macrophages. This latter effect was mediated by the glucocorticoid receptor, which led to decreased chemokine secretion and expression. In vivo results were quite comparable; treatment of high fat diet-fed mice with dexamethasone prevented ATM accumulation in epididymal fat. This decrease in ATM was most pronounced for the proinflammatory F4/80+, CD11b+, CD11c+ M-1-like ATM subset. Overall, our results elucidate a beneficial function of peroxisome proliferator-activated receptor γ activation and glucocorticoid receptor/glucocorticoids in adipose tissue and indicate that pharmacologic prevention of ATM accumulation could be beneficial.

Keratinocyte-derived Chemokine in Obesity: EXPRESSION, REGULATION, AND ROLE IN ADIPOSE MACROPHAGE INFILTRATION AND GLUCOSE HOMEOSTASIS*

Obese adipose tissue (AT) is associated with chronic inflammation, and we hypothesized that the keratinocyte-derived chemokine (KC), the mouse ortholog of human interleukin-8, plays a role in obesity-mediated AT inflammation and the subsequent manifestation of insulin resistance. KC expression is increased in the AT and plasma of genetically (ob/ob) and high fat diet-induced obese mouse models, and this increase may be mediated by the elevated leptin and tumor necrosis factor-α levels associated with obesity. Obesity-induced KC expression occurs primarily in stromal vascular cells and not in adipocytes, and it is high in preadipocytes and decreases during adipogenesis. Although KC has no effect on adipogenesis, it induces adipocyte expression of inflammatory factors and the insulin resistance mediator, suppressor of cytokine signaling 3. Using chimeric mice deficient in the KC receptor CXCR2 in their bone marrow, we show that the lack of CXCR2 in hematopoietic cells is sufficient to protect from adipose and skeletal muscle macrophage recruitment and development of insulin resistance in diet-induced obese mice. These studies suggest that KC and its receptor CXCR2 are potential targets for the development of new therapeutic approaches for treatment of obesity-related insulin resistance, type II diabetes, and related cardiovascular diseases.

Ligand-receptor interactions of the low density lipoprotein receptor-related protein, a multi-ligand endocytic receptor

Summary The low density lipoprotein receptor-related protein (LRP) is a large membrane glycoprotein that is a member of the low density lipoprotein (LDL) receptor family of endocytic receptors. In contrast to the restricted ligand specificity of the LDL receptor, LRP can bind and internalize a remarkable spectrum of structurally-unrelated classes of ligands suggesting a role for the receptor in diverse physiological and patho-physiological processes ranging from lipoprotein metabolism, cell growth and cell migration to atherosclerosis and Alzheimers disease. In this review we will summarize the current insights in the biology of LRP and particularly focus on the recent progress in our understanding of the molecular mechanisms that enable LRP to interact specifically with such a multitude of different ligands.

Interaction between factor VIII and LDL receptor-related protein. Modulation of coagulation?

Recent reports suggest that the multifunctional receptor low-density lipoprotein receptor-related protein (LRP) may contribute to the regulation of blood coagulation by mechanisms that differ from the simple removal of protease/inhibitor complexes from the circulation. This possibility became apparent from the observation that LRP is involved in down-regulation of Tissue Factor expression at the surface of monocytes and fibroblasts. Furthermore, coagulation Factor VIII and activated Factor IX (Factor IXa) have been identified as proteins that are able to bind to LRP. In the present review, the potential contribution of LRP to the regulation of the coagulation cascade through these novel pathways is discussed, with particular reference to the interaction between LRP and coagulation Factor VIII.

Blockade of α4 integrin Signaling Ameliorates the Metabolic Consequences of High Fat Diet-Induced Obesity

OBJECTIVE—Many prevalent diseases of advanced societies, such as obesity-induced type 2 diabetes, are linked to indolent mononuclear cell–dependent inflammation. We previously proposed that blockade of α4 integrin signaling can inhibit inflammation while limiting mechanism-based toxicities of loss of α4 function. Thus, we hypothesized that mice bearing an α4(Y991A) mutation, which blocks signaling, would be protected from development of high-fat diet–induced insulin resistance. RESEARCH DESIGN AND METHODS—Six- to eight-week-old wild-type and α4(Y991A) C57Bl/6 male mice were placed on either a high-fat diet that derived 60% calories from lipids or a chow diet. Metabolic testing was performed after 16–22 weeks of diet. RESULTS—α4(Y991A) mice were protected from development of high-fat diet–induced insulin resistance. This protection was conferred on wild-type mice by α4(Y991A) bone marrow transplantation. In the reverse experiment, wild-type bone marrow renders high-fat diet–fed α4(Y991A) acceptor animals insulin resistant. Furthermore, fat-fed α4(Y991A) mice showed a dramatic reduction of monocyte/macrophages in adipose tissue. This reduction was due to reduced monocyte/macrophage migration rather than reduced monocyte chemoattractant protein-1 production. CONCLUSIONS—α4 integrins contribute to the development of HFD-induced insulin resistance by mediating the trafficking of monocytes into adipose tissue; hence, blockade of α4 integrin signaling can prevent the development of obesity-induced insulin resistance.

The second and fourth cluster of class a cysteinerich repeats of the low density lipoprotein receptor-related protein (LRP) share ligandbinding properties

The low density lipoprotein receptor-related protein (LRP) is a multifunctional endocytic cell-surface receptor that binds and internalizes a diverse array of ligands. The receptor contains four putative ligand-binding domains, generally referred to as clusters I, II, III, and IV. In this study, soluble recombinant receptor fragments, representing each of the four individual clusters, were used to map the binding sites of a set of structurally and functionally distinct ligands. Using surface plasmon resonance, we studied the binding of these fragments to methylamine-activated alpha(2)-macroglobulin, pro-urokinase-type plasminogen activator, tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1, t-PA.plasminogen activator inhibitor-1 complexes, lipoprotein lipase, apolipoprotein E, tissue factor pathway inhibitor, lactoferrin, the light chain of blood coagulation factor VIII, and the intracellular chaperone receptor-associated protein (RAP). No binding of the cluster I fragment to any of the tested ligands was observed. The cluster III fragment only bound to the anti-LRP monoclonal antibody alpha(2)MRalpha3 and weakly to RAP. Except for t-PA, we found that each of the ligands tested binds both to cluster II and to cluster IV. The affinity rate constants of ligand binding to clusters II and IV and to LRP were measured, showing that clusters II and IV display only minor differences in ligand-binding kinetics. Furthermore, we demonstrate that the subdomains C3-C7 of cluster II are essential for binding of ligands and that this segment partially overlaps with a RAP-binding site on cluster II. Finally, we show that one RAP molecule can bind to different clusters simultaneously, supporting a model in which RAP binding to LRP induces a conformational change in the receptor that is incompatible with ligand binding.