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Diabetes Publish Ahead of Print, published online April 21, 2008 Integrin signaling and insulin resistance Blockade of α4 integrin Signaling Ameliorates the Metabolic Consequences of High Fat Diet-Induced Obesity Chloé C. Féral1,2§ PhD, Jaap G. Neels1 PhD, Christiane Kummer1 PhD, Marina Slepak1 BS, Jerrold M. Olefsky1 MD PhD, and Mark H. Ginsberg1 MD 1Department of Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0726, 2INSERM, U634, Nice F-06107, France, Nice-Sophia Antipolis University. 28 avenue de Valombrose, Nice cedex 2. § To whom correspondence should be addressed. Email [email protected]. Received for publication 13 December 2007 and accepted in revised form 14 April 2008. Additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org Copyright American Diabetes Association, Inc., 2008 Integrin signaling and insulin resistance 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 (HFD)-induced insulin resistance. Research Design and Methods: 6-8 weeks old wild-type (WT) and α4(Y991A) C57Bl/6 male mice were placed on either a HFD that derived 60% calories from lipids, or a chow diet. Metabolic testing was performed after 16 to 22 weeks of diet. Results: α4(Y991A) mice were protected from development of HFD-induced insulin resistance. This protection was conferred on WT mice by α4(Y991A) bone marrow transplantation. In the reverse experiment, WT bone marrow renders HFD-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 (MCP- 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. 2 Integrin signaling and insulin resistance besity leads to insulin resistance that α4 integrin signaling, to perturb functions results in Type 2 Diabetes (1) and that involved in inflammation, while limiting O contributes to hypertension and mechanism-based adverse effects (14). α4 cardiovascular disease (2). Mononuclear cell- integrin signaling involves the binding of mediated inflammation in obese adipose paxillin to the α4 integrin tail and a point tissue plays a pathogenetic role in insulin mutation (α4Y991A) that selectively blocks resistance (3; 4). Thus, there is great interest this interaction reduces α4-mediated in the possibility of using anti-inflammatory leukocyte migration (15) and adhesion- strategies to ameliorate obesity-induced strengthening in flowing blood (16), while insulin resistance. sparing α4-mediated static cell adhesion (17). Blockade of leukocyte adhesion is a Furthermore, mice bearing an α4(Y991A) proven therapeutic strategy for a wide variety mutation are viable and fertile and have intact of inflammatory diseases (5). In particular, lympho-hematopoiesis and humoral immune inhibiting α4 integrins or their counter- responses; however, they exhibit defective receptors (VCAM-1 and MadCAM-1) blocks recruitment of mononuclear leukocytes in inflammatory responses mediated by experimental inflammation (18). Here we mononuclear leukocytes (6). Indeed α4 report that the α4(Y991A) mutation reduces integrin antagonists are of proven benefit in mononuclear leukocyte infiltration of white several human inflammatory diseases (7; 8). adipose tissue in high fat diet (HFD)-induced These antagonists, such as the monoclonal obese mice, and hence reduce HFD-induced antibody natalizumab, block ligand binding insulin resistance. Thus, we establish that function, thus producing a complete loss of α4 blocking α4 integrin signaling can ameliorate integrin function. Lack of α4 integrins is the metabolic consequences of HFD-induced embryonic lethal and results in defective obesity. placentation, heart development, and hematopoiesis (9-11). Furthermore, RESEARCH DESIGN AND METHODS natalizumab therapy has been associated with fatal progressive multifocal Animals and animal care. The α4(Y991A) leukoencephalopathy in humans, possibly due mice were previously described and have to defective T cell trafficking to the brain (12; been backcrossed 9 times onto the C57BL/6 13). Thus, currently available α4 integrin background (18). We fed male mice (aged 6-8 antagonists are of proven value in weeks) either on HFD, containing 60% fat by mononuclear cell-mediated diseases; however weight (D12492, Research Diets Inc.) or on complete loss of α4 integrin function is chow diet (10% fat, D12450B, Research Diets associated with developmental defects and Inc.) for 16 to 22 weeks. All experiments abnormal hematopoiesis. were approved by the UCSD IACUC. As noted above, whereas α4 integrin antagonists show promise for several Glucose and insulin tolerance test. We carried autoimmune and inflammatory diseases, out GTTs, and ITTs as described (19) (see mechanism-based toxicities may limit their Supplementary Methods). use, particularly in low grade chronic inflammatory conditions such as obesity- Whole blood and plasma measurements. Total induced insulin resistance. We recently white blood cell number and differential proposed an alternative strategy, blockade of counts were assessed by standard techniques 3 Integrin signaling and insulin resistance (ACP Diagnostic Lab, UCSD). We measured allowed 4 weeks for reconstitution of donor plasma insulin by radioimmunoassay (Linco marrow, which we verified by PCR (18). Research) and determined FFAs by colorimetric assay (Wako). Plasma cytokines Isolation of stromal vascular (SV) cells. were measured by the core laboratories of the Epidydimal fat pads were minced in PBS and Diabetes and Endocrinology Research digested with collagenase (Roche) and Consortium (UCLA, LINCOplex assay for DNAse I (Sigma) for 1h at 37°C degrees. Mouse Cytokines, Suspensions were then filtered through http://www.derc.med.ucla.edu/core.htm). 100µm nylon cell strainer then spun at 1000g Plasma cholesterol and triglyceride levels for 10 minutes. Preadipocyte/adipocyte were measured by enzymatic methods using enriched fraction was removed. Isolated SV an automated bichromatic analyzer (Abbot cells were resuspended in RBC lysis buffer Diagnostics, USA). All these measurements (KHCO3 10mM, NH4Cl 150mM, EDTA pH8 were performed on 11 WT and 9 α4(Y991A) 0.1mM) for 5 minutes at RT. Cell suspension mice for HFD, and 6 WT and 5 α4(Y991A) was centrifuged 5 min at 500g and SV cells for chow diet (Table 1). were resuspended in PBS at 106 cells per 100µl. Histochemistry. Adipose tissue was fixed overnight in 10% formaldehyde, dehydrated in ethanol bath and paraffin-embedded. Flow Cytometry. Cells were harvested from Sections were stained with hematoxylin and white adipose tissue, peripheral blood and eosin for observation of adipose tissue bone marrow, and incubated in Fc blocker (rat structure. anti-mouse CD16/32) for 20 min at room temperature. The cells were then incubated Pancreas isolation and determination of islet with FITC-Ly-6G (1/200) and PE-7/4 (1/50) area. Pancreata were isolated, and fixed in (BD Biosciences/PharMingen (San Diego, 4% formalin over night. Paraffin sections CA). Negative control staining was were generated and stained with H&E. performed with FITC-rat IgG2a/k and PE- rat Pictures were taken of H&E stained pancreas IgG2a. Cell staining was analyzed with sections and the area of Langerhans islets as FACScan flow cytometer using CellQuest well as the total area of the pancreas were software (BD Biosciences Systems, CA). measured using ImageJ software (NIH freeware). Islet area was depicted as % of Cell migration assay. Cell migration was total pancreas area. Sections of 4-7 mice per assayed in a modified Boyden chamber group and at least 6 different pancreas areas system using 24 well-transwell plate (8µm per mouse were analyzed for statistical pore size, Corning), coated with 5µg/ml significance using unpaired 2-tailed Student’s VCAM-1 (R&D systems). MCP1 (R&D t test. P values less than 0.05 were considered systems) was added in the lower chamber at significant. 1nM. Cells harvested from WT or α4(Y991A) bone marrow were grown for 5-7 days in the Bone marrow transplantation. We injected presence of GM-CSF (5 µl of 0.1mg/ml stock 4 bone marrow obtained from WT and solution). BM-derived macrophages (2x10 ) α4(Y991A) mice (4x106 cells) through the tail were kept in suspension in 1% serum vein into male C57BL/6 (4 months) and containing medium for 1 hour at RT. Cells α4(Y991A)(4–6 months) mice that had been were then added to the top chamber and irradiated (10 Gy) 4 hours before. Mice were incubated overnight at 37°C. Filters were 4 Integrin signaling and insulin resistance fixed, stained with crystal violet and migrated pancreatic β cells than those bearing the cells in the lower chamber were enumerated. α4(Y991A) mutation (Fig.1C) in response to the HFD. Since increased β cells is an early RNA isolation and RT-PCR.