RAGE Regulates the Metabolic and Inflammatory Response to High-Fat

1948 Diabetes Volume 63, June 2014 Fei Song,1 Carmen Hurtado del Pozo,1 Rosa Rosario,1 Yu Shan Zou,1 Radha Ananthakrishnan,1 Xiaoyuan Xu,2 Payal R. Patel,3 Vivian M. Benoit,3 Shi Fang Yan,1 Huilin Li,4 Richard A. Friedman,5 Jason K. Kim,3 Ravichandran Ramasamy,1 Anthony W. Ferrante Jr.,2 and Ann Marie Schmidt1 RAGE Regulates the Metabolic and Inflammatory Response to High-Fat Feeding in Mice Diabetes 2014;63:1948–1965 | DOI: 10.2337/db13-1636 In mammals, changes in the metabolic state, including The constellation of obesity, insulin resistance, and di- obesity, fasting, cold challenge, and high-fat diets abetes results from the integration of metabolic and (HFDs), activate complex immune responses. In many inflammatory signals. When imbalances in caloric intake strains of rodents, HFDs induce a rapid systemic and energy expenditure contribute to obesity, disordered inflammatory response and lead to obesity. Little is cross-talk among adipocytes, liver, brain, and skeletal known about the molecular signals required for HFD- muscle emerges, thereby eliciting cues that result in induced phenotypes. We studied the function of the tissue recruitment of inflammatory cells. A consequence of receptor for advanced glycation end products (RAGE) inflammatory cell recruitment to key metabolic organs, in the development of phenotypes associated with particularly macrophage populations to visceral adipose high-fat feeding in mice. RAGE is highly expressed on tissue, is the derangement of the insulin signaling pathway, immune cells, including macrophages. We found that leading to impaired glucose and lipid homeostasis (1–5). high-fat feeding induced expression of RAGE ligand In this context, we hypothesized that receptor for HMGB1 and carboxymethyllysine-advanced glycation advanced glycation end products (RAGE) might contrib- end product epitopes in liver and adipose tissue. OBESITY STUDIES Genetic deficiency of RAGE prevented the effects of ute to the pathogenesis of obesity and insulin resistance HFD on energy expenditure, weight gain, adipose induced by a high-fat diet (HFD). RAGE is a multiligand fl tissue inflammation, and insulin resistance. RAGE receptor that recognizes stress and in ammatory signals, deficiency had no effect on genetic forms of obesity high mobility group box 1 (HMGB1), advanced glycation caused by impaired melanocortin signaling. Hemato- end products (AGEs), S100/calgranulins, lysophosphatidic – poietic deficiency of RAGE or treatment with soluble acid, phosphatidyl serine, and ITGAM (6 11). RAGE is RAGE partially protected against peripheral HFD- highly expressed on monocytes and macrophages, and induced inflammation and weight gain. These findings its expression is further enhanced after immune activa- demonstrate that high-fat feeding induces peripheral tion or infection (12–16). Although seminal roles for inflammation and weight gain in a RAGE-dependent RAGE in carbohydrate excess have been well studied, manner, providing a foothold in the pathways that the regulation of RAGE signaling in the response to regulate diet-induced obesity and offering the potential disturbances in lipid metabolism has been less well for therapeutic intervention. characterized. 1Diabetes Research Program, Division of Endocrinology, Department of Medicine, Corresponding authors: Anthony W. Ferrante Jr., [email protected], and Ann New York University School of Medicine, New York, NY Marie Schmidt, [email protected]. 2 Naomi Berrie Diabetes Center, Columbia University College of Physicians and Received 22 October 2013 and accepted 3 February 2014. Surgeons, New York, NY This article contains Supplementary Data online at http://diabetes 3Program in Molecular Medicine and Division of Endocrinology, Diabetes and .diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1636/-/DC1. Metabolism, Department of Medicine, University of Massachusetts Medical School, Worcester, MA F.S. and C.H.d.P. contributed equally to this work. 4Departments of Population Health (Biostatistics) and Environmental Medicine, A.W.F. and A.M.S. contributed equally to this work. New York University School of Medicine, New York, NY © 2014 by the American Diabetes Association. See http://creativecommons.org 5Biomedical Informatics Shared Resource, Herbert Irving Comprehensive Cancer /licenses/by-nc-nd/3.0/ for details. Center and Department of Biomedical Informatics, College of Physicians and Surgeons, Columbia University, New York, NY diabetes.diabetesjournals.org Song and Associates 1949 High-fat feeding induces an inflammatory response in conscious mice using a primed and continuous infusion the liver, adipose tissue, and hypothalamus (17–19). The of human insulin (Humulin; Eli Lilly and Company, Indi- regulators of this inflammatory response remain largely anapolis, IN) at a rate of 15 pmol/kg/min to raise plasma obscure. We tested the hypothesis that high-fat feeding insulin levels to within a physiologic range. Blood samples triggers production and accumulation of RAGE ligands in (20 mL) were collected at 20-min intervals for the imme- key metabolic tissues, which activate inflammatory signal- diate measurement of plasma glucose concentration; 20% ing leading to obesity and insulin resistance. To test this glucose was infused at variable rates throughout the 2-h model, we measured two key proinflammatory RAGE experiment to maintain plasma glucose at basal concen- ligands in metabolic tissues and the effects of preventing trations. Basal and insulin-stimulated whole-body glucose RAGE activation in mice fed HFD. High-fat feeding indu- turnover was assessed with continuous infusion of [3-3H] ces RAGE ligand Hmgb1 mRNA transcripts and carboxy- glucose (PerkinElmer Life and Analytical Sciences, Boston, methyllysine (CML)-AGE epitopes in liver and adipose MA) before (0.05 mCi/min) and throughout the clamps tissue, and inhibition of RAGE by genetic or pharmaco- (0.1 mCi/min). To estimate insulin-stimulated glucose logic means has led to the unexpected result in mice that uptake in individual organs, 2-deoxy-D-[1-14C] glucose RAGE is required for the development of diet-induced (2[14C]DG) (PerkinElmer) was administered as a bolus obesity and its associated pathologies of insulin resistance (10 mCi) 75 min after the start of the clamps. Blood and dyslipidemia. Furthermore, RAGE in bone marrow– samples were taken during and at the end of the clamps fi 23 3 derived cells accounts, at least in part, for these ndings. for measurement of plasma [3 H] glucose, H2O, and The present data reveal fundamental metabolic sensing 2[14C]DG concentrations and insulin. At the end of the roles for RAGE and suggest that blockade of RAGE may clamp, mice were killed, and the organs were collected for prevent diet-induced obesity and metabolic dysfunction. multiple biochemical analyses, including measures of gly- colysis and glucose production as previously described RESEARCH DESIGN AND METHODS (24). Animals and Diets 2 2 Measurement of Glucose, Lipid, Nonesterified Fatty Homozygous RAGE null mice (C57BL/6 Ager / mice Acids, and Insulin Levels in Fasted Mice backcrossed .20 generations into C57BL/6J [The Jackson Blood glucose was measured with a FreeStyle blood Laboratory, Bar Harbor, ME]) and their littermate RAGE- glucose meter and strips (Abbott Diabetes Care Inc., expressing controls were used. Ay mice (B6.Cg-Ay/J) (20) Alameda, CA). Plasma cholesterol concentrations were were purchased from The Jackson Laboratory and bred measured with standard assays (Infinity Cholesterol Re- with homozygous RAGE null mice to generate C57BL/6 2 2 agent; Fisher Diagnostics, Middletown, VA) or (Cholesterol Ay/a; Ager / and littermate controls. All mice studied E; Wako Diagnostics, Richmond, VA). The concentration of were male, had free access to water, and were subjected serum nonesterified fatty acids was measured using to 12-h light/dark cycles. At 6–8 weeks of age, mice were a colorimetric assay NEFA C test kit (Wako Diagnostics). fed HFD with 60% of calories from lard (D12492; Re- Blood insulin concentrations were measured by ELISA search Diets, Inc., New Brunswick, NJ) or low-fat diet (ALPCO Diagnostics, Salem, NH) (Fig. 8D) or by rat ul- (LFD) with 13% of calories from fat (5053 PicoLab Rodent trasensitive ELISA (cross-reacts with murine insulin) (Fig. Diet 20; LabDiet, Brentwood, MO). Fat and lean masses 2B) (Mercodia Inc., Winston-Salem, NC). were measured by dual-energy X-ray absorptiometry (DEXA) scanning. All animal procedures were approved Glucose and Insulin Tolerance Tests by the Columbia University, New York University, and After baseline collection of blood, glucose 2 g/kg or insulin University of Massachusetts Medical School Institutional (Eli Lilly and Company) 0.75 units/kg was injected Animal Care and Use Committees and performed in ac- intraperitoneally. Serial blood glucose analysis was per- cordance with the National Institutes of Health Animal formed through 120 min postinjection (25). Care Guidelines. Soluble RAGE (sRAGE) (human) was prepared, puri- fied, and rendered free of endotoxin (16), and 100 mg/day Energy Balance and Hyperinsulinemic-Euglycemic i.p. was administered to mice. Vehicle consisted of equal Clamp Studies volumes of PBS. Metabolic studies were performed at the National Mouse Metabolic Phenotyping Center at UMass. Metabolic cages Real-Time Quantitative PCR (TSE Systems, Inc., Midland, MI) were used in conscious Total RNA was extracted from liver or epididymal fat mice to simultaneously measure energy expenditure tissue using the RNeasy lipid kit (Qiagen, Hilden, Germany). through

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