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Theses & Dissertations Boston University Theses & Dissertations

2014

Role of the Receptor for Advanced

Glycation End products (RAGE) in

adipose tissue: browning effect

https://hdl.handle.net/2144/14372

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BOSTON UNIVERSITY SCHOOL OF MEDICINE

Thesis

ROLE OF THE RECEPTOR FOR ADVANCED GLYCATION END PRODUCTS (RAGE) IN ADIPOSE TISSUE: BROWNING EFFECT

by

CYNTHIA SHIM

B.A., New York University, 2011

Submitted in partial fulfillment of the requirements for the degree of

Master of Science 2014

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© 2014 by

CYNTHIA SHIM All rights reserved

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Approved by

First Reader

Barbara Seaton, Ph.D.

Professor of Physiology and Biophysics

Second Reader

Ann Marie Schmidt, M.D.

Principal Investigator, Diabetes Research Program New York University School of Medicine

Third Reader

Carmen Hurtado del Pozo, Ph.D.

Postdoctoral Fellow, Diabetes Research Program

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ROLE OF THE RECEPTOR FOR ADVANCED GLYCATION END-PRODUCTS (RAGE) IN ADIPOSE TISSUE: BROWNING EFFECT

CYNTHIA SHIM ABSTRACT

Adipose tissue plays an essential role in the regulation of many metabolic processes. Excess caloric intake and decreased energy expenditure cause adipocyte hypertrophy and hyperplasia, leading to inflammation of the adipose tissue, which contributes to obesity. Recent data link the Receptor for Advanced Glycation End-products (RAGE) to high fat diet (HFD)-induced obesity and subsequent metabolic dysfunction, but its function is incompletely understood. On HFD, the concentration of RAGE ligands, such as carboxy methyl lysine (CML) – advanced glycation end-products (AGE) epitopes, S100 calcium binding protein B (S100b), and high mobility group box 1 (HMGB1) increases, activating RAGE and resulting in inflammation. However, deletion of RAGE protected against the HFD-induced obesity and resulted in increased overall energy expenditure. In this study, we tested the hypothesis that the protective mechanism of RAGE deletion is due, in part, to browning which involves inducing brown adipose tissue (BAT)-like properties in white adipose tissue (WAT). The expression of

uncoupling protein 1 (UCP1), which is usually only expressed in BAT, is increased in the WAT of RAGE knockout (RKO) mice. This effect was reproduced in vitro, by silencing the Ager gene in the adipocyte cell line C3H10T1/2. We propose that RAGE affects adipocyte phenotype by downregulating the expression of browning genes such as UCP1, and therefore is a key determinant of energy expenditure and adiposity. Thus, RAGE

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antagonism may promote BAT phenotype and function in WAT, reducing adiposity, which may have potential therapeutic implications for treating obesity.

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TABLE OF CONTENTS

TITLE………...i

COPYRIGHT PAGE………...ii

READER APPROVAL PAGE………..iii

ABSTRACT ... iv  

TABLE OF CONTENTS ... vi  

LIST OF TABLES ... vii  

LIST OF FIGURES ... viii  

LIST OF ABBREVIATIONS ... ix  

INTRODUCTION ... 1  

Adipose Tissue ... 2  

RAGE ... 8  

Hypothesis: Current Study ... 10  

MATERIALS AND METHODS ... 12  

RESULTS ... 17  

DISCUSSION ... 25  

REFERENCES ... 28  

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LIST OF TABLES

Table Title Page

1 Characteristics of WAT and BAT 5

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LIST OF FIGURES

Figure Title Page

1 Energy Balance 2

2 Adipose Tissue Biology 3

3 Adipocyte-Immune Cell Interactions 4

4 Locations of BAT 6

5 Activators of BAT 8

6 Variants of RAGE and an Overview of Key Post-receptor Events Following Full-length RAGE Activation

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7 RAGE Expression in Different Fat Depots 17

8 HFD increases PGAT accumulation of Hmgb1 and CML 18

9 RAGE null mice are resistant to HFD induced obesity 20

10 Markers of browning 21

11 C3H10T1/2 cell line as a model of adipogenesis 22

12 RAGE inhibits browning 24

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LIST OF ABBREVIATIONS

Actb ... β-actin AGE ... Advanced Glycation End-products

Ager ... Advanced glycosylation end product-specific receptor ATP ... Adenosine Triphosphate BAT ... Brown Adipose Tissue BMI ... Body Mass Index

Cidea ... Cell death-inducing DNA fragmentation factor, alpha subunit-like effector A CML ... Carboxy Methyl Lysine CVD ... Cardiovascular Disease DMEM/F12 ... Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 FA ... Fatty Acid

Fasn ... Fatty acid synthase FDG ... Fluoro-labeled 2-Deoxyglucose HFD ... High Fat Diet HMGB1 ... High Mobility Group Box 1

Hmgb1 ... High mobility group box 1 ICL ... Innate Lymphoid Cell LFD ... Low Fat Diet

Lipe ... Lipase, hormone sensitive NAFLD ... Non-Alcoholic Fatty Liver Disease NCS ... Newborn Calf Serum

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PBS ... Phosphate-Buffered Saline PGAT ... Perigonadal Adipose Tissue

Pnpla2 ... Patatin-like phospholipase domain containing 2

Ppargc1a ... Peroxisome proliferative activated receptor, gamma, coactivator 1 alpha RAGE ... Receptor for Advanced Glycation End-products RKO ... RAGE Knockout S100b ... S100 calcium binding protein B SCAT ... Subcutaneous Adipose Tissue SDS-PAGE ... Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

Srebf1 ... Sterol regulatory element binding transcription factor 1

Tfam ... Transcription factor A, mitochondrial TG ... Triglyceride TIID ... Type 2 Diabetes Treg ... Regulatory T cell UCP1 ... Uncoupling Protein 1 WAT ... White Adipose Tissue WT ... Wild Type

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INTRODUCTION

During the past 20 years there has been a dramatic increase in obesity in the United States and rates remain high. Overweight and obesity are both classifications for ranges of weight that are greater than what is generally considered healthy for a given height. For adults, overweight and obesity ranges are determined by calculating a number called the body mass index (BMI) which correlates with amount of body fat. An adult with a BMI between 25 and 29.9 is considered overweight. An adult with a BMI greater than 30 is considered obese. The most recent national data on obesity prevalence show that more than one-third of U.S. adults (35.7%) are obese (Center for Disease Control and Prevention, 2012). Obesity is closely related to many disorders, including type 2 diabetes mellitus, insulin resistance, dyslipidemia, and cardiovascular diseases (Malnick &

Knobler, 2006). Diet, genetic predisposition factors and lifestyle influence both energy intake and expenditure levels (Figure 1). Obesity develops when energy intake exceeds energy expenditure. According to Tseng et al. (2010), a small portion of nutrient energy intake is lost in feces and urine; a portion is used for physiological needs; a variable portion is used in physical activity; while the majority is used for metabolic processes or is lost in the production of heat. Of the remaining energy loss, there is the heat produced during digestion and absorption of food (thermic effect of food), the thermic effect of exercise, and the energy dissipated in response to the environmental changes, such as cold temperature and diet. These latter forms of regulated heat production are referred to as adaptive thermogenesis, and occur primarily in the mitochondria of skeletal muscle

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and brown fat; it is finely regulated and therefore has the potential to be manipulated therapeutically to serve as a target for obesity treatment (Tseng et al., 2010). While most current obesity therapies are focused on reducing caloric intake, recent data suggest that increasing cellular energy expenditure (bioenergetics) may be an attractive alternative approach (Tseng et al., 2010).

Figure 1. Energy Balance. When the human body’s energy balance is shifted towards expenditure (out), there is less inflammation and lower risk of disease. However, when there is more energy in than out, the human body may become resistant to insulin and more susceptible to diseases. Abbreviations: FA (fatty acids); ATP (adenosine

triphosphate); TG (triglycerides); NAFLD (non-alcoholic fatty liver disease); TIID (type 2 diabetes); CVD (cardiovascular disease). Figure amended from Birerdinc et al., 2013.

Adipose Tissue

Until the 1940s, adipose tissue was described as a type of connective tissue that contained lipid droplets. However, this slowly began to change as research showed that

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adipose tissue is the site of calorie storage after feeding and is the source of circulating fatty acids during fasting (Rosen & Spiegelman, 2014). In the last decade, adipose tissue has become indisputably recognized as an active endocrine organ, with the discovery of adipose-derived cytokines, such as leptin, that act upon other major organs. The

importance of adipose tissue as an endocrine organ along with the enormous increase in global obesity rates sparked a renewed interest in adipose tissue (Figure 2).

Figure 2. Adipose Tissue Biology. Blue represents the total number of adipose papers when the term ‘adipose’ was searched in PubMed. Red represents adipose papers as a percentage of all papers. Important findings are indicated by publication year. Figure taken from Rosen & Spiegelman, 2014.

One of the exclusive characteristics of adipose tissue is its ability to change its dimensions. During overnutrition, adipose depots expand first by increasing the size of individual cells (hypertrophy) until a critical threshold of about 0.8 µg/cell is reached

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(Krotkiewski et al., 1983). If overnutrition continues, there is an increased need to store excess calories. Thus, signals are released to recruit new adipocytes (hyperplasia) from the resident pool of progenitors by inducing their proliferation and/or differentiation (adipogenesis). In addition to the matrix of extracellular proteins that holds the adipose depots together, adipocytes are surrounded by a wide variety of cells that includes endothelial cells, fibroblasts, stem cells, and immune cells. In lean animals, tissue-remodeling/wound healing, anti-inflammatory M2 macrophages dominate the adipose tissue resident population (Figure 3). However, in a continued state of overnutrition, more pro-inflammatory M1 macrophages infiltrate the adipose depot along with

neutrophils, mast cells, B and T lymphocytes, etc., contributing to insulin resistance (Oh et al., 2012).

Figure 3. Adipocyte-Immune Cell Interactions. The immune cell population in the lean adipose depot is predominated by M2 macrophages, eosinophils and regulatory T cells (Tregs). When overnutrition occurs, there is an accumulation of proinflammatory cells, including M1 macrophages, mast cells, and various T lymphocytes. Abbreviations: ICL (innate lymphoid cell). Figure taken from Rosen & Spiegelman, 2014.

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Due to the association of adipose tissue with metabolic disease, the benefits of healthy adipose tissue are often overlooked. Besides its importance in energy

homeostasis, adipose tissue also has important mechanical qualities. It protects delicate organs such as the eyes, and cushions body parts exposed to high levels of mechanical stress such as the heels and toes. It also plays an important role in providing thermal insulation (Rosen & Spiegelman, 2014). The most important role of adipose tissue by far is in regulation of energy balance and nutritional homeostasis.

There are two main types of adipose tissue – WAT (white adipose tissue) and BAT (brown adipose tissue). The two types of WAT we studied are the visceral

perigonadal adipose tissue (PGAT) and the inguinal subcutaneous adipose tissue (SCAT). The main function of WAT is to store excess energy in the form of triglycerides, whereas the BAT is specialized to oxidize fatty acids and dissipate energy as heat through UCP1 (uncoupling protein 1)-mediated uncoupling of oxidative phosphorylation from ATP synthesis. White adipocytes are generally larger in size than brown adipocytes. White adipocytes are characterized by a single large lipid droplet, whereas brown adipocytes contain numerous small droplets. In addition, WAT has fewer mitochondria in

comparison to BAT. Another important difference between the two is that WAT does not express UCP1. Under normal conditions, UCP1 expression is exclusive to BAT. Other major differences between WAT and BAT are listed in Table 1 (Birerdinc et al., 2013). Table 1. Characteristics of WAT and BAT. This table lists the major differences between WAT and BAT. Table amended from Birerdinc et al., 2013.

White adipose tissue Brown adipose tissue Cells are 70-80 nm in diameter Cells are 30‒40 μm in diameter Cells contain a single large droplet Cells contain numerous smaller droplets

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Few mitochondria Relatively large number of mitochondria Low iron content Abundant iron

Enriched in monoenic acids Enriched in free cholesterol and phospholipids Contains substantial amounts of linoleic acid Little vascularization/capillaries Abundant vascularization/capillaries Number of WAT cells positively correlates with insulin

resistance

Number of BAT cells negatively correlates with insulin resistance

No UCP1 expression Express UCP1 Do not produce heat Produce heat

Prior to 2007, BAT was considered to be nonexistent or nonfunctional in adult humans and only present in infants. However, the study by Saito et al. (2009), together with three recent reports in the New England Journal of Medicine (Cypess et al., 2009; van Marken Lichtenbelt et al., 2009; Virtanen et al., 2009), demonstrates that healthy adult humans have significant depots of metabolically active BAT. Positron emission tomography is commonly used to detect highly metabolically active cancer cells based on their uptake of large amounts of fluoro-labeled 2-deoxyglucose (FDG). During these imaging studies, localized depots of fatty tissue were frequently identified as hot spots for FDG uptake. Specifically, tissue biopsies corresponding to the positron emission

tomography–positive regions have the morphological and molecular characteristics of BAT, including expression of the BAT-specific protein UCP1.

Figure 4. Locations of BAT. Examples of brown adipose tissue in a newborn (A) (Image taken from Merklin, 1974); a 13-year-old boy (B) (Image taken from Hong et al., 2011), an adult male (C) (Image taken from van Marken Lichtenbelt et al., 2002) and a morbidly obese male (D) (Image taken from Vijgen et al., 2011).

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Recently, it was discovered that when some depots of WAT were subjected to certain stimuli (Figure 5) such as cold exposure, they could adopt a BAT phenotype, expressing a core program of thermogenic genes, including UCP1 (Wu et al., 2012). These inducible adipocytes are known as beige adipocytes. Beige adipocytes have low thermogenesis activity and a small number of mitochondria like white adipocytes, but when they are activated, they possess many BAT-like features, such as the presence of multilocular lipid droplets, abundant mitochondria, and UCP1 expression (Lo & Sun, 2013). This suggests that beige adipocytes are programmed to be bifunctional – suited for energy storage in the absence of thermogenic stimuli, but fully capable of turning on heat production in the presence of appropriate signals (Wu et al., 2012). Browning of WAT has been shown to have anti-obesity effects in rodent models (Wu et al., 2013).

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Figure 5. Activators of BAT. Many activators of browning and thermogenesis have been discovered. Figure taken from Rosen & Spiegelman, 2014.

RAGE

RAGE (Receptor for Advanced Glycation End-products) is a pattern recognition receptor that recognizes stress and inflammatory signals (Schmidt et al., 1992). RAGE is highly expressed on many immune cells and its expression is further enhanced when immune activation occurs. RAGE is also highly expressed on a variety of cells, including adipocytes (Schmidt et al, 1992). RAGE binds AGEs (advanced glycation end-products), which form during irreversible post-translational modification of proteins, lipids or nucleic acids. For example, glycolysis results in the formation of a number of reactive products that have the potential to interact with protein residues to rapidly form AGEs

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(Barlovic et al., 2011). In addition to binding with AGEs, RAGE interacts with CML-AGE epitopes (carboxy methyl lysine – advanced glycation end-products), S100b (S100 calcium binding protein B), and HMGB1 (high mobility group box 1) (Schmidt et al., 2000). The interaction between RAGE and these ligands released by inflamed, stressed and damaged cells is thought to result in pro-inflammatory gene expression. Increased expression of both RAGE and accumulation of its ligands are observed in a range of disorders characterized by chronic inflammation, such as rheumatoid arthritis,

atherosclerosis, Alzheimer’s disease and the vascular complications of diabetes (Bierhaus et al., 2005).

RAGE is composed of a large extracellular domain, a single transmembrane domain, and a 43-amino-acid cytoplasmic tail (Dattilo et al., 2007). The ligand binding site has been identified to be in the V-type immunoglobulin-like extracellular domain, whereas the cytosolic tail mediates intracellular signaling (Barlovic et al., 2011). Splice variants of murine and human RAGE have also been identified, including soluble

isoforms, whose roles are not yet completely understood (Kalea et al, 2009). The RAGE-ligand interaction triggers activation of NF-κB and other signaling pathways (Figure 6). Increased expression of inflammatory cytokines follows, leading to an inflammatory response (Hofmann et al., 1999). In obesity, the combined effects of overnutrition, low-energy expenditure, hyperglycemia and hyperlipidemia may augment the formation of AGEs that activate RAGE, producing inflammatory mediators that contribute to the development of obesity-associated complications (Gaens et al., 2013).

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Figure 6. Variants of RAGE and an Overview of Key Post-receptor Events Following Full-length RAGE Activation. By triggering the NF-κB pathway, activation of RAGE triggers an inflammatory response. Figure amended from Barlovic et al., 2011.

Hypothesis: Current Study

RAGE plays a key role in adipocytes by contributing to high fat diet-induced obesity and its subsequent complications. Mice that are deficient in RAGE are resistant to high fat diet-induced obesity and also show increased energy expenditure and higher body temperatures (Song et al., 2014). It was in this context that we hypothesized that

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there is a link between RAGE and regulation of UCP1, perhaps by the browning of WAT.

We tested the hypothesis that the reduced adiposity, increased energy expenditure and higher body temperature observed in RAGE null mice are a result of browning of WAT. To test our model in vivo, we assessed the expression levels of UCP1 in WAT of RAGE null mice compared with wild type (WT) mice. To test our hypothesis in vitro, we silenced RAGE in an adipocyte cell line and compared our findings to the appropriate control treatments. Our experiments show that UCP1 expression increases in the absence of RAGE. These findings reveal RAGE and its ligands as possible regulators of the browning of WATs and suggest that this browning may contribute to the protective mechanism of RAGE deficiency. This would have important medical implications because a BAT-like phenotype is associated with lower risk of obesity, making it a potential target for treating obesity.

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MATERIALS AND METHODS

Animals and diets

Homozygous RAGE null mice (C57BL/6 Ager -/- mice backcrossed >20 generations into C57BL/6J (Jackson Laboratory, Bar Harbor ME)) and their littermate RAGE-expressing WT controls were used. All mice studied were male, had free access to food and water, and were subjected to 12-hour light/dark cycles. At 6 to 8 weeks of age, mice were fed HFD with 60% of calories from lard (diet D12492; Research Diets Inc., New Brunswick, NJ) or low fat diet (LFD) with 13% of calories from fat (5053; PicoLab Rodent Diet 20; Lab Diet, Brentwood, MO).

Primary cultures of white adipocytes

PGAT from WT and RAGE null mice (RKO) were excised, rinsed in phosphate-buffered saline (PBS), minced and treated with collagenase for 45 minutes to 1 hour at 37°C with shaking. The digestion was stopped by adding pre-warmed high glucose Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12). Cell

suspensions were filtered through a 100 µm filter and then centrifuged at 1000 rpm for 4 minutes. The pellet was resuspended, filtered through a 40 µm filter and centrifuged at 2000rpm for 10 minutes. Precursor cells were seeded to obtain 1500–2000 cells/cm2 on day 1 and grown in DMEM supplemented with 10% newborn calf serum (NCS), 3 nm insulin, 10 mm HEPES, 50 IU penicillin and 50 µg streptomycin/ml, and 15

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Precursor cells proliferated actively in these conditions, reached confluence on days 4–5 after seeding (40,000–80,000 cells/cm2), and were fully differentiated by day 8 into mature white adipocytes. Cells were used to check the expression on RAGE at different days of differentiation.

Preparation of C3H10T1/2 cells

A vial of C3H10T1/2 cells was taken out of the dry ice tank and held in a 37°C bath to defrost. Cells were mixed into complete medium and centrifuged for 7 minutes at room temperature. The pellet was resuspended into complete medium and placed in a 37°C incubator. The cells grew to about 80% confluence and were detached from the flask by adding trypsin and incubating for two minutes. The cells were centrifuged for 7 minutes and the pellet was resuspended and either kept in complete medium at 37°C or frozen for future use. Cells were grown to confluence in DMEM, 10% (vol/vol) FBS and 1% (vol/vol) penicillin-streptomycin followed by standard adipogenic induction (20nM insulin, 1nM T3, 0.5 mM isobutylmethylxanthine, 1µM dexamethasone, 0.125 mM indomethacin) and stimulation with a prototypic AGE prepared in our laboratory, carboxy methyl lysine (CML) AGE or vehicle. RNA and protein from mature cells were prepared. After a total of 12 days of incubation, cells exhibited a fully differentiated phenotype with substantial lipid droplet accumulation.

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24 hours prior to transfection, the medium was changed to Optimem.

Undifferentiated C3H10T1/2 cells were transfected with Ambion Silencer RAGE siRNA or scramble control using lipofectamine and stored at 37°C. The next day, the medium was changed back to complete medium to allow the cells to recover for at least 24 hours in the incubator, before differentiation.

Quantitative Real-Time PCR analysis

Tissue samples were homogenized in QIAzol reagent using a homogenizer. Total RNA from either homogenized tissues or differentiated C3H10T1/2 cells was isolated using RNeasy Mini columns following the mantufacturer’s instructions. cDNA was synthesized with multiscribe reverse transcriptase (Applied Biosystems, Foster City, CA). 5µl of cDNA was used in a 20µl quantitative PCR reaction using the TaqMan method (50°C for 2 minutes, 95°C for 10 minutes, and 40 cycles of 95°C for 15 seconds and 60°C for 1 minute) with premade primer sets (Applied Biosystems). PCR reactions were run in duplicate for each sample. The relative abundance of transcripts was normalized according to the expression of β-actin using the ΔΔCt method and mRNA expression was quantified using Microsoft Excel.

Table 2. Target genes. This table lists the genes that were searched in our real-time PCR experiments.

Gene Name Gene Symbol Assay ID Advanced glycosylation end product-specific receptor Ager Mm01134790_g1

β-actin Actb Mm00607939_s1

Cell death-inducing DNA fragmentation factor, alpha subunit-like effector A Cidea Mm00432554_m1 Fatty acid synthase Fasn Mm00662319_m1 High mobility group box 1 Hmgb1 Mm00849805_gH Lipase, hormone sensitive Lipe Mm00495359_m1

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Peroxisome proliferative activated receptor, gamma, coactivator 1 alpha Ppargc1a Mm01208835_m1 Patatin-like phospholipase domain containing 2 Pnpla2 Mm00503040_m1 Sterol regulatory element binding transcription factor 1 Srebf1 Mm00550338_m1 Transcription factor A, mitochondrial Tfam Mm00447485_m1 Uncoupling protein 1 (mitochondrial, proton carrier) Ucp1 Mm01244861_m1

Western Blot analysis

Tissue samples were homogenized in lysis buffer using a homogenizer and differentiated C3H10T1/2 cells were placed in lysis buffer. After lysis, lysates were purified by centrifugation at 11,000 rpm for 15 minutes at 4°C. The supernatants were isolated and protein concentration was quantified using the Thermo Scientific Pierce BCA Protein Assay kit. Proteins were separated by sodium dodecyl

sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose

membranes. Membranes were blocked for 20 minutes at room temperature to reduce non-specific antibody binding. Then, the membranes were incubated with the primary

antibodies overnight at 4°C. After triple-washing the membranes to remove unbound primary antibody, membranes were incubated with secondary antibodies for one hour at room temperature. Membranes were triple-washed again and the presence of specific proteins was visualized using Odyssey. Protein expression levels were quantified using Image J and Microsoft Excel.

Statistical Analyses

All data are reported as mean ± standard error of the mean. Statistical significance between two groups was determined by two-tailed Student’s t-test using Microsoft Excel.

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Statistical significance between four groups was determined with a one-way ANOVA followed by an unpaired, two-tailed student’s t-test for P value using IBM SPSS Advanced Statistics 20.0. A p value of 0.05 or less was considered statistically significant.  

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RESULTS

To study the possible role of RAGE in specific adipose tissue stores, we analyzed its mRNA expression and protein levels in different fat depots of LFD-fed wild-type mice. RAGE expression was higher in interscapular BAT than the visceral white PGAT and the inguinal white SCAT (Figure 7A and B). These results suggested that RAGE might contribute to energy expenditure regulation and to obesity.

Figure 7. RAGE expression in different fat depots. Two months after induction of LFD, PGAT, SCAT and BAT were extracted and subjected to (A) real time quantitative PCR for detection of Ager mRNA transcripts (N=5 mice/group) and (B) Western blot analysis for expression of RAGE in protein (N=3 mice/group).

To determine if RAGE plays a role in the inflammatory and metabolic responses to high fat feeding, we assessed the concentrations of three prototypical proinflammatory RAGE ligands – CML AGE, S100b and Hmgb1. Two months after the start of LFD or HFD, PGAT, SCAT and BAT were retrieved and subjected to either real-time

quantitative PCR for detection of Hmgb1 mRNA transcripts or Western blotting for detection of CML and S100b. The concentrations of both Hmgb1 mRNA transcripts and CML-AGE were significantly higher in the PGAT of mice fed HFD than in mice fed

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LFD (Figure 8A and B). In addition, Western blots for expression of S100b showed an increase of S100b in all adipose tissue depots of HFD mice (Figure 8C). Expression of RAGE was also significantly higher in HFD mice (Figure 8D).

Figure 8. HFD increases PGAT accumulation of Hmgb1 and CML. In mice fed HFD vs. LFD, PGAT was retrieved after two months of diet and subjected to real time quantitative PCR for detection of Hmgb1 mRNA transcripts (A) and Western blotting for detection of CML (B) (N=5 mice/group) and S100b (C) N=3 mice/group. After incubation of the blots with primary antibody, blots were stripped and re-probed with anti-β-actin IgG. Analysis of intensity of CML bands were normalized to β-actin. LFD and HFD adipocytes were subjected to real time quantitative PCR for detection of Ager mRNA transcripts (D).

In experiments performed previously by other members of the laboratory, RKO mice on HFD showed higher temperatures and increased energy expenditure when

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compared with its WT counterparts (Figure 9A and B). RAGE null mice were protected from the weight gain seen in WT mice fed HFD (Figure 9C). These data suggested that RAGE deficiency protects against effects of HFD on local adipose tissue inflammation and insulin resistance, possibly due, in part, to the increased body temperature.

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Figure 9. RAGE null mice are resistant to HFD induced obesity. WT or RKO mice were placed on either LFD or HFD. Body temperature was monitored (A) N=6 mice/group. Mice were placed in metabolic chambers for assessment of VO2 (B) and VCO2 (C) at baseline, 3 weeks HFD and 6 weeks HFD, N=6 mice/group. Body mass was measured (D) N=7-8 mice/group. * indicates p<0.001 comparing WT vs. RKO mice fed HFD. Figures taken from Song et al., 2014.

These findings prompted us to assess the levels of the thermogenic gene Ucp1 in BAT, PGAT and SCAT of WT vs. RKO mice on HFD. Ucp1 mRNA transcripts were significantly higher in PGAT of RKO vs. WT mice on HFD (Figure 10A), which suggests that RAGE deficiency induces a BAT gene program in WAT of HFD mice. To further characterize adipose tissue in the presence or absence of RAGE, we measured mRNA expression of known BAT markers including PGC-1α (encoded by Ppargc1a), cell-death activator (Cidea), and transcription factor A (Tfam). Expression of all BAT marker genes was significantly increased in the visceral white PGAT, with Tfam being the only exception (Figure 10B). These data indicate that RAGE deficiency increases BAT marker expression in visceral WAT.

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Figure 10. Markers of browning. BAT, PGAT and SCAT were retrieved for WT and RKO mice fed LFD and HFD and subjected to real time quantitative PCR for Ucp1

expression (A). PGAT from WT and RKO mice fed LFD or HFD were retrieved and subjected to real-time quantitative PCR for detection of Ppargc1a, Cidea and Tfam

mRNA transcripts (B).

To further investigate our findings of the increased uncoupling in RKO WAT, we used the C3H10T1/2 cell line, which is an established model of adipogenesis. There was a similar pattern of RAGE expression in primary preadipocytes obtained from mice and C3H10T1/2 – derived adipocytes, which demonstrates that the C3H10T1/2 cell line is an effective model of adipogenesis (Figure 11A and B). Photos taken before and after C3H10T1/2 cell differentiation show the accumulation of lipid droplets by day 12 of differentiation (Figures C and D).

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Figure 11. C3H10T1/2 cell line as a model of adipogenesis. Compared to primary preadipocyte differentiation (A), adipocyte differentiation of the C3H10T1/2 cell line shows similar patterns of expression for RAGE. Photos were taken of the C3H10T1/2 cells before differentiation (C) and after 12 days of differentiation (D). Both photos are taken at 40x magnification.

To mimic deletion of RAGE in primary adipocytes, we silenced RAGE

expression via short interfering (si)RNA transfection prior to differentiation. There was no difference in the expression of genes responsible for lipolysis (Pnpla2, Lipe) and lipogenesis (Fasn, Srebf1) in siRAGE cells as compared to the cells transfected with a scrambled sequence of the siRNA target sequence, i.e., negative control. As expected, silencing RAGE in these C3H10T1/2 cells significantly induced Ucp1 gene and protein expression as compared to the negative control (Figure 12A and B). This supports an experiment done in our lab previously that showed that when RAGE antibody was used to block RAGE in the cell line C3H10T1/2, UCP1 expression increased (Figure 12C). These results collectively suggest that RAGE expression suppresses browning gene expression programs such as UCP1. In addition, our results show that when RAGE is blocked, browning increases.

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Figure 12. RAGE inhibits browning. C3H10T1/2 cells with RAGE silenced (siRAGE) and their controls (scramble) were subjected to Western blotting (A) and real-time quantitative PCR (B) for detection of UCP1 (p<0.05). When compared to RAGE antibody-blocked C3H10T1/2 cells (C), both methods of stimulating RAGE deficiency show similar results.

   

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DISCUSSION

Given its central role in obesity and subsequent complications, adipose tissue has become an important therapeutic target. Inducing BAT-like characteristics in WAT may be a therapeutic strategy for reducing adiposity. In mice, the metabolic rate of BAT is nearly two orders of magnitude higher than that in any other tissue (Cannon and Nedergaard, 2004). Rothwell and Stock (1979) calculated that 40 – 50 grams of BAT could account for 20% of daily energy expenditure, which would be astounding, given that there are at least 100 grams of BAT in a healthy adult person (van Marken

Lichtenbelt et al., 2009). However, it is necessary to gain a better understanding of the pathways that promote and inhibit beige adipocyte thermogenesis in humans.

Previous research from our laboratory implicated important roles for RAGE expression in adipocytes and perhaps other cells such as macrophages by contributing to diet-induced obesity and subsequent metabolic dysfunction via activating inflammatory pathways. In comparison with their WT counterparts, RKO mice on HFD were shown to be resistant to obesity, have higher body temperatures, and increase energy expenditure. The goal of the current study was to determine if the protective mechanism of RAGE deletion is due to browning of WAT. To study the role of RAGE, 2 to 3 month old WT and RKO mice on LFD and HFD were used to visualize the effects on browning gene expression programs, particularly UCP1. Additionally, C3H10T1/2 cell lines were used to test if our results are consistent in vitro.

The WT mice were first examined to illustrate the normal expression patterns of RAGE in adipose tissue. During LFD, RAGE showed an adipose depot-specific

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expression pattern in WT mice, with expression highest in BAT and lowest in PGAT. During HFD, results showed that the absence or deficiency of RAGE increased

expression of UCP1 and other classical BAT markers in PGAT, with minimal changes in SCAT and BAT. These results suggested a link between RAGE and browning. Our findings show that altering RAGE and/or its ligands can promote functional plasticity of adipocytes, and identify RAGE as a possible determinant of white vs. brown fat

transformation. The possibility that other RAGE effects or UCP1-independent pathways might contribute to thermogenesis in RAGE deficiency cannot be ignored. Similarly, the specific mechanisms through which RAGE-ligand interactions induce a BAT-like thermogenic program remain to be clarified.

Increased clearance of energy stores by brown and beige adipocytes could reduce the excess of triglycerides and glucose associated with obesity. Moreover, it could produce beneficial metabolic effects or protection from obesity. However, whether nutrients absorbed by beige adipocytes are used in nonshivering thermogenesis, as they are in classic brown adipocytes, remains unclear. Despite these uncertainties, the impact of RAGE on body temperature and energy expenditure in obese mice identifies RAGE as a novel therapeutic target in the treatment of obesity.

Here we establish RAGE and its ligands as previously unidentified determinants of functional browning of WAT. From our findings, the presence of RAGE and its ligands suppresses browning. Therefore, deleting or blocking RAGE can activate a thermogenic program, promoting energy dissipation and limiting obesity. To date, no definitive therapy prevents the consequences of HFD and diminished energy expenditure.

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Disruption of RAGE expression or function in visceral white fat could offer new opportunities in targeting and treating obesity and its associated complications.

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