The attenuating effects of pyridoxamine on adipocyte hypertrophy and inflammation differ by adipocyte location

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ScienceDirect Journal of Nutritional Biochemistry 72 (2019) 108173

The attenuating effects of pyridoxamine on adipocyte hypertrophy and inflammation differ by adipocyte location☆ Seyeon Oh a , Hyosang Ahn a, b , Hyunjin Park a, b , Jae-Ik Lee c , Kook Yang Park c , Daehee Hwang d, Sojung Lee a, b , Kuk Hui Son c,⁎, Kyunghee Byun a, b,⁎⁎ a

Functional Cellular Networks Laboratory, Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Incheon, Republic of Korea b Department of Anatomy and Cell Biology, Graduate school of Medicine, Gachon University, Incheon, 21936, Republic of Korea c Department of Thoracic and Cardiovascular Surgery, Gachon University Gil Medical Center, Gachon University, Incheon, Republic of Korea d Department of New Biology and Center for Plant Aging Research, Institute for Basic Science, Daegu Gyeongbuk Institute of Science and Technology, Daegu 42988, Republic of Korea

Received 1 April 2018; received in revised form 19 October 2018; accepted 4 April 2019

Abstract It is known that receptor for advanced glycation end products (RAGE) and its ligands accumulate in the fat tissues of obese individuals, and RAGE ligands induce M1 macrophage polarization, which in turn induces inflammation. We evaluated the effect of pyridoxamine on RAGE ligand accumulation and M1 polarization in the visceral, subcutaneous, and perivascular fat tissues of Sprague-Dawley rats fed a high fat diet (HFD). Pyridoxamine reduced HFD-induced weight gain, attenuated adipocyte size increases, RAGE ligand accumulations, RAGE-RAGE ligands binding, decreased macrophage M1 polarization and increased M2 polarization in visceral fat tissues, but not in subcutaneous tissues. Pyridoxamine induced glyoxalase 1 (Glo-1) expression in visceral fat in the HFD group, whereas pyridoxamine induced Glo-1 expression in perivascular fat tissues was no higher than that observed in the normal fat diet (NFD) controls. In vitro, pyridoxamine suppressed the release of RAGE ligands from AGE treated macrophages, but non-significantly attenuated RAGE ligands release in AGE treated adipocytes. Pyridoxamine was found to suppress weight increases and M1 polarization, and to increase Glo-1 expression through the RAGE pathway in perivascular and visceral fat tissues of HFD-induced obese rats. These findings suggest pyridoxamine is a candidate for the treatment of obesity or complications related to obesity-induced inflammation. © 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Keywords: Obesity; Fat; Receptor for advanced glycation end products; Macrophage polarization; Inflammation; Pyridoxamine

1. Introduction In the obese, visceral fat tissue produces more inflammatory cytokines and is more infiltrated by inflammatory cells (predominantly macrophages) than subcutaneous fat tissues [1,2], which is why visceral fat tissue is more related to obesity-induced diseases, such as, cardiovascular disease and diabetes, than subcutaneous fat tissue [1,2]. Recently perivascular fat has attracted much research attention [3]. This tissue type is particularly susceptible to inflammation, as evidenced by the fact that levels of monocyte chemoattractant protein (MCP-1; a pro-inflammatory chemokine that induces macrophage recruitment and activation) are more than 40-fold higher in perivascular adipocytes than subcutaneous adipocytes [4].

Receptor for advanced glycation end products (RAGE) has several ligands, such as, advanced glycation end products (AGEs), members of the proinflammatory S100/calgranulin family, and high motility group box 1 protein (HMGB-1) [5,6]. After binding these ligands, RAGE activates inflammation-related signaling cascades involving nuclear factor-(NF)κB, ERK (extracellular signal-regulated kinase) 1/2, p38 MAPK (mitogen-activated protein kinases), JNK (c-Jun N terminal kinases), PKC (protein kinase C), Rac/Cdc42, or TIRAP and MyD88 (adaptor proteins for TLR 2 and 4) [5,6]. These observations regarding the potential regulatory role of RAGE in inflammation suggest RAGE might importantly contribute to inflammation and to the obesity-associated dysregulation of adipokines [7]. Several studies have shown RAGE is related to obesity. In particular, RAGE expression was found to be up-regulated in the

☆ This research was supported by the Basic Science Research Program through the National Research Foundation of Korea [grant number, 2016R1D1A1B03933680, KH]; and the Jeisys of Korea [grant number, 20175274, KB]. ⁎ Correspondence to: K.H. Son, 1198 Guwall-dong, Namdong-gu, Incheon 21565, Republic of Korea. Tel.: +82 32 899 6511; fax: +82 32 899 6519. E-mail addresses: [email protected] (K.H. Son), [email protected] (K. Byun).

https://doi.org/10.1016/j.jnutbio.2019.04.001 0955-2863/© 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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adipocytes of obese humans [7], and RAGE ligands, such as, AGEs, HMGB, and S100/calgranulins have been reported to accumulate in high fat diet (HFD) and other models of obesity [8,9]. RAGE ligands also cause macrophage polarization, which is frequently observed in the visceral fat tissues of obese individuals. M1 polarized macrophages are pro-inflammatory tissue-destructive macrophages, whereas M2 macrophages are anti-inflammatory and mediate tissue repair [10]. When primary murine bone marrow derived macrophages were incubated with AGE, proportions of M1 polarized macrophages were observed to increase in a RAGE-dependent manner [11]. In addition, it was reported that genetic RAGE deficiency blocks the effects of a HFD on energy expenditure, weight gain, adipose tissue inflammation, and insulin resistance [8]. Pyridoxamine is a highly potent AGE inhibitor of considerable research interest. It is a vitamin B6 analog and an anti-glycating agent [12,13], and has been reported to significantly suppress the upregulations of proinflammatory genes in the visceral adipose tissues of HFD mice [12]. However, these studies were largely conducted on visceral adipocytes in vitro models based on human adipocyte cells or on visceral adipocytes in the HFD animal models not in the perivascular fat tissue. We hypothesized pyridoxamine action on the adipocytes might differ in visceral, subcutaneous, and perivascular fat tissues, because inflammation induction by adipocytes through RAGE pathway differ among those fat tissue. In this study, we compared the effects on pyridoxamine action on adipocyte area, RAGE ligand accumulation, and M1/M2 polarization in visceral, subcutaneous, and perivascular fat tissues.

differentiate in maintenance medium (DMEM containing 10% FBS) for 9 days (the medium was changed every 2 days). 3T3-L1-MBX cells were incubated with 800 ng/ml AGE treatment with or without 400 ng/ml pyridoxamine for 2 days. All cells were maintained in a 5% CO2 humidified incubator at 37°C.

2.2.3. Proliferation assay The 3T3-L1-MBX were seeded in 96-well plates at 1 × 104 cells (SPL life sciences, Gyeonggi-do, Korea). After 24 h of cell culture, rinsed 2 times with PBS and then, the cells were treated with reagents and compounds for 24 h. After 24 h, the reagents were removed and cells were rinsed 2 times with PBS. Then the cells were treated with mixed serum free medium and Transdetect cell counting kit solution (10:1 ratio, v:v; TransGen Biotech, Beijing, China). After 4 h, absorbance was measured by microplate reader (Spectra max plus).

2.3. Sample preparation 2.3.1. Tissues for staining Rats were euthanized with 10 mg/kg of Rumpun (Bayer Korea, Ansan, Korea) and 500 mg/kg of Zoletil (BK Pharm, Ilsan, Korea). Subcutaneous fat tissues were collected from shoulders and chest walls, visceral fat from omentum and mesentery, and perivascular fat from around aortas. Tissues were fixed in 4% paraformaldehyde (P2031, Biosesang, Seongnam, Korea) overnight at 4°C and then placed in an automatic dehydration machine (ASP300S, Leica, Milton Keynes, UK). Tissues were dehydrated in 90% ethanol for 3×1 hour, 100% ethanol for 2×2 h, cleared with 100% xylene 3×1.5 h, and then embedded in paraffin.

2.3.2. Protein for ELISA and Western blot Subcutaneous, visceral, and perivascular fat proteins were extracted using the EzRIPA lysis kit (WSE-7420, ATTO, Tokyo, Japan). Initially, tissues were homogenized with lysis buffer containing proteinase and phosphatase inhibitors and briefly sonicated for 10×10 seconds in a cold bath sonicator. After centrifuging at 14,000×g for 20 minutes at 4°C, supernatants were collected and protein concentrations were determined using a Bicinchoninic acid assay kit (BCA assay kit, 23227, Thermo Fisher, IL, USA).

2. Materials and methods 2.1. Animals The Sprague-Dawley rats (8 weeks) used in this study were maintained in a temperature-controlled room (24°C) under a 12 h light-dark cycle. The study was approved by the Lee Gil Ya Cancer and Diabetes Institute of Gachon University, and was conducted in accordance with the guidelines issued by our Institutional Animal Care and Use Committee (AAALAC International; approval number; LCDI–2016-0006). One week after arrival, rats were divided into three groups and fed either a 45% high-fat diet (D12451, Research Diets, New Brunswick, NJ, USA) or a normal diet (the HFD and NFD groups, n=6, respectively) for 8 weeks (Supplementary Fig. 1). Rats in the pyridoxamine (P9380, Sigma-Aldrich, MO, USA) treated group was fed a HFD for 4 weeks and then pyridoxamine (2 mg; the dose was as determined by Raval et al. [14]) prepared by dissolution in 1 ml of saline was orally administered daily for another 4 weeks with feeding a 45% high-fat diet (the HFD/PM group, n=6). After these periods, animals were sacrificed. Body weights were measured daily. We measured AGE level in the diet by AGE ELISA kit (MBS700464, MyBioSource, SD, USA). The concentration of AGEs in HFD was 5.16±1.25 mg/ml and that of NFD was 4.23±0.53 mg/ml. The concentration of AGEs in HFD was higher than in NFD, but this was not statistically significant. 2.2. Cell culture 2.2.1. Macrophages Murine macrophages (Raw 264.7) were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM, SH30081.01, Hyclone, UT, USA) supplemented with 10% fetal bovine serum (FBS, TMS-013-BKR, Merck, NJ, USA), 100 U/ml penicillin and 100 μg/ml streptomycin (P/S, 15-140-148, Gibco, CA, USA). Raw 264.7 cells were incubated with 800 μg/ml AGE (A8301, Sigma-Aldrich, MO, USA) with or without 400 ng/ml pyridoxamine at 37°C in a 5% CO2 atmosphere for 2 days.

2.3.3. RNA and cDNA Tissues were homogenized in ice using a disposable pestle in 1 ml of TRIzol (12183555, Invitrogen, CA, USA), and then added to 0.2 ml of chloroform (0757, Amresco, OH, USA), mixed, and centrifuged at 12,000×g for 15 minutes at 4°C. Aqueous phases were collected, placed in cleaned tubes, mixed with 0.5 ml of isopropanol, and centrifuged using the same conditions. Isolated RNA was then washed with 70% ethanol, and dissolved in 30 μl of DEPC treated water. To perform qRT-PCR (quantitative realtime polymerase chain reaction), cDNA was synthesized from 1 μg of total RNA using a PrimeScript 1st strand cDNA Synthesis Kit (#6110A, TAKARA, Shiga, Japan).

2.3.4. Serum For preparation serum, collected blood was incubated at room temperature for 20 minutes. After 20 min, blood was centrifuged at 3,000 rpm for 10 minutes at 4°C. And then, blood was aliquoted supernatant. We have used that supernatant (serum).

2.4. Triglyceride measurements Blood samples were collected when fat tissues were harvested, and serum triglyceride levels were measured using Triglyceride Quantification kits (ab65336, Abcam, Cambridge, UK), according to the manufacturer’s instructions.

2.5. Total cholesterol, glucose measurements The obtained transparent serum specimens were stored in a freezer at -80°C until the clinical chemistry test (KPNT, Gyunggi-do, Korea). The selected analyses were total cholesterol and glucose of serum level.

2.6. Histological assessments 2.2.2. Adipocyte cells Mus musculus pre-adipocyte cells (3T3-L1-MBX) were purchased from the Korean Cell Line Bank and the ATCC and cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM, LM001-05, WELLGENE, Gyeongsan, Korea) supplemented with 10% fetal bovine calf serum (FBS, TMS-013-BKR, Merck, NJ, USA), 100 U/ml penicillin and 100 μg/ml streptomycin and cultured at 37°C in a 5% CO2 atmosphere until confluent. Cells were differentiated 2 days after reaching confluence by culturing them in DMEM containing 10% FBS, 1 μg/mL insulin (I0516, Sigma-Aldrich, MO, USA), 1 μM dexamethasone (API-04, G Biosciences, MO, USA) and 0.5 mM methylisobutylxanthine (IBMX, I5879, Sigma-Aldrich, MO, USA) for 2 days, and then grown in high glucose DMEM containing only insulin for another 2 days. Subsequently, cells were allowed to

For histologic analysis, paraffin embedded fat tissues were sectioned at 5 μm and stained with hematoxylin (s3309, DAKO, Tokyo, Japan) and eosin (318906, SigmaAldrich, MO, USA). Microphotographs were obtained using an Axio Imager Z1 upright microscopy system at x20 (Carl Zeiss, Oberkochen, Germany). Adipocyte area was measured the cross-sectional area of adipocyte cellular membranes. To measure this area, ImageJ 1.50i software was used to quantify the cross-sectional are. The number of 60 adipocytes were selected randomly from one image of fat tissues. Each selected adipocyte was measured the length of edge and these measurements from one image were averaged. Brown fat in subcutaneous, visceral and perivascular fat was distinguished from white fat by its smaller lipid droplets and color [15].

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2.7. Enzyme-linked immunosorbent assay (ELISA) assay 2.7.1. Quantitative ELISA assay Absolute concentrations of RAGE ligands and RAGE in cells, serum and tissue samples were determined using ELISA kits (AGE, MBS700464, MyBioSource, SD, USA; HMGB1, MBS703437, MyBioSource, SD, USA; S100β, MBS702860, MyBioSource, SD, USA; RAGE, ab202409, Abcam, Cambridge, UK). Briefly, standards and samples in 96-well plates were incubated for 2 h at 37°C for RAGE ligands and for 1 hour at room temperature for RAGE. After removing liquid, biotin antibodies were incubated for 1 hour at 37°C. Unbound antibodies were removed by washing with Wash Buffer, and then incubated with HRP-avidin for 1 hour at 37°C. After washing out unbound HRP-avidin, color was developed by incubating samples with TMB Substrate during for 15-30 minutes at 37°C for RAGE ligands and for 10 minutes at room temperature for RAGE. The reaction was stopped by adding Stop solution to wells. Absorbance were measured at 450 nm using an ELISA plate reader (Molecular Devices, VERSAMAX, CA, USA).

2.7.2. Sandwich ELISA assay Interactions between RAGE and RAGE ligands in fat tissues were investigated by sandwich ELISA. A 96-well plate was coated with anti-RAGE antibody in coating solution mixture overnight at 4°C. Unbound anti-RAGE antibody was removed with washing with TPBS. To reduce non-specific binding, plates were treated with 5% skim milk containing TPBS and incubated overnight at 4°C. Unbound proteins were removed by washing repeatedly with TPBS. Anti-RAGE antibody binding samples were treated with anti-AGE, anti-HMGB1, and anti-S100β antibodies for 2 h at room temperature. After washing with TPBS, bound proteins were incubated with HRP conjugated antirabbit secondary antibody for 2 h at room temperature. After washing off unbound HRP

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conjugated secondary antibody, color was developed by incubating samples with TMB for 15 minutes. The reaction was stopped by adding 100 μl of 2 M H2SO4 to each well, and absorbance were then measured at 450 nm using an ELISA plate reader (Molecular Devices, VERSAMAX, CA, USA).

2.8. Western blot Protein obtained in RIPA lysis buffer (ATTO, Tokyo, Japan) and EasySee protein marker (TransGen Biotech Co., LTD, Beijing, China) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to polyvinypdylidene fluoride (PVDF) membranes using Semi-Dry at 25V for 10 mins (ATTO, Tokyo, Japan). Transferred membranes were blocked with 5%(w/v) skimmed milk in Tris-buffered saline (pH 7.6) (TBS) containing 0.1% Tween-20 (TBST) for 1 h. After washing, membranes were incubated with primary antibodies (listed in Supplementary Table 1) in blocking solution overnight at 4°C, washed with TBST, incubated with appropriate secondary antibodies, and rewashed. Proteins were detected using ECL star (Dyne bio, Gyeonggi-do, Korea) on a LAS-4000 (GE Healthcare, IL, USA).

2.9. Immunohistochemistry (3,3-diaminobenzidine; DAB) The fat paraffin tissue slides were removed paraffin and incubated in normal animal serum to block antibody binding, incubated with RAGE antibody (sc-365154, Santa Cruz, CA, USA) for 2 days at 4°C, and then rinsed twice with PBS. They were then treated with biotinylated secondary antibodies in the ABC kit (PK-6101, Vector Laboratories, CA, USA), incubated for 1 hour in blocking solution, and rinsed twice with PBS. Finally,

Fig. 1. Relationships between body weight, triglyceride level, and adipocyte size in the high fat diet and pyridoxamine treated rats. (A, B) Mean body weight in the high fat diet pyridoxamine treated (HFD/PM) group was less than in the high fat diet (HFD) group, although but mean weight gain in HFD/PM group was greater than in the normal fat diet (NFD) group. (C, D, E) However, no difference was observed between serum triglyceride, total cholesterol and glucose levels in the NFD and HFD/PM groups. Serum triglyceride levels were higher in the HFD group than in the HFD/PM group. (F, G) Hematoxylin and eosin stained fat tissues showed that adipocytes were larger in subcutaneous and visceral fat tissues in the HFD and HFD/PM groups than in the NFD group, but adipocytes were smaller in visceral fat tissues of the HFD/PM group than that of HFD group. (H) Mean adipocyte size ratio in perivascular fat tissue was higher in the HFD group than in the HFD/PM group. (200× magnification). NFD: normal fat diet, HFD: high fat diet, HFD/PM: high fat diet plus pyridoxamine, SQ: subcutaneous fat, VIS: visceral fat, PV: perivascular fat. Results are presented as means±SDs. Scale bar=50 μm; *, ** and ***, significantly different (Pb.05, Pb.01 and Pb.001) from the NFD group; $, $$ and $$$, statistical significantly different (Pb.05, Pb.01 and Pb.001) from the HFD group.

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sections were reacted with 3,3'-diaminobenzidine (DAB, D5637, Sigma-Aldrich, MO, USA) substrate for few minutes. 2.10. Immunofluorescence (IF) The fat paraffin tissue slides were removed paraffin and incubated in normal animal serum to block antibody binding, incubated with antibodies (listed in Supplementary Table 1) for 3 days at 4°C, and then rinsed twice with PBS. It was incubated for 1 hour in in fluorescence conjugated secondary antibody, and then rinsed 3 times with PBS. Then it was incubated for 5 mins in DAPI solution (1 μg/ml) and then rinsed 3 times with PBS. Finally, it was mounted with cover slip and vector shield solution (Vector Laboratories). Then fluorescence was detected a confocal microscope (LSM 710; Carl Zeiss, Oberkochen, Germany). 2.11. Quantitative real-time polymerase chain reaction (qRT-PCR) qRT-PCR was used to determine levels of Cd86 and Cd206(surrogates of M1 and M2 macrophage polarization), Glo-1 and NF-κB, TNF-α and IL-1β (inflammation factors). Briefly, 100 ng of cDNA, 5 μl of ROX plus SYBR green premix (4367659, Thermo Fisher, IL, USA) and 0.4 μM of forward and of reverse primers (Supplementary Table 2) were mixed, and levels of gene expression were then determined using a CFX384 Touch™ Real-Time PCR Detection System (6096, Bio-Rad, CA, USA). Gene expression levels were normalized with respect to the Actb gene and then normalized versus to NFD group of each type fat. 2.12. Statistical analysis Given the small sample size, non-parametric analysis was used. The Kruskal-Wallis test was used to compare groups, and the Mann-Whitney U test was used for multiple comparisons. Statistical analyses were performed using SPSS version 22 (IBM Corporation, NY, USA), and P values of b.05 were deemed significant. Results are expressed as means±standard deviations.

the HFD and HFD/PM groups. However, in visceral and perivascular fat tissues, the mRNA levels of as Pparγ, Cebpa and Srebps were lower in the HFD/PM group than in the HFD group, and levels in the HFD/PM group were higher than in the NFD group (Supplementary Fig. 2A-C). In subcutaneous fat tissues, the mRNA levels of lipogenesis factor such as Fabp, Lpl, and Hsl [18–20] were higher in the HFD and HFD/PM groups than in the NFD group, and those levels were similar in the HFD and HFD/PM groups. However, in visceral and perivascular fat tissues the mRNA levels of Fabp, Lpl, and Hsl were lower in the HFD/PM group than in the HFD group, and levels in the HFD/PM group were higher than in the NFD group (Supplementary Fig. 2D-F). 3.3. Pyridoxamine affected the accumulations of RAGE ligands in fat tissues The accumulations of RAGE ligands, that is, AGE, AGE-albumin, HMGB1, and S100β, in fat tissues were investigated by ELISA (Fig. 2AD, ***P=.000, vs NFD; $$$, P=.000, vs HFD). In subcutaneous fat, these 4 ligands accumulated significantly more in the HFD and HFD/PM groups than in the NFD group, whereas levels in the HFD and HFD/PM groups were not significantly different. In addition, ligand accumulations were significantly lower in the HFD/PM group than in the HFD group, but significantly higher in the HFD/PM group than in the NFD group in visceral and perivascular fat. Immunofluorescence stains of AGE, HMGB1, and S100β showed same patterns with in direct ELISA data (Supplementary Fig. 3). 3.4. Pyridoxamine affected the serum levels of RAGE ligands

3. Results 3.1. Pyridoxamine reduced weight gain and triglyceride increases after 4 weeks on the HFD Animals in the HFD group gained weight significantly more than animals in the NFD group (Fig. 1A). Animals in the HFD group were fed only the HFD, whereas animals in the HFD with pyridoxamine group (the HFD/PM group) were fed PM and HFD, and animals in the NFD group were fed a normal diet for one more month. Weight gain was significantly less in the HFD/PM group than in the HFD group after 2 months (Fig. 1B, ***P=.000 or 0.000, vs NFD; $$, P=.008, vs HFD) although weight gain in the HFD/PM group was greater than in the NFD group. Mean triglyceride level, total cholesterol, glucose level in the HFD/PM group was significantly lower than in the HFD group, and similar to that observed in the NFD group (Fig. 1C-E). 3.2. Pyridoxamine reduced the adipocyte area increase induced by HFD Mean white adipocyte area in subcutaneous fat in the HFD and HFD/PM groups were significantly greater than in the NFD group, but no difference was observed between white adipocyte area in the HFD and HFD/PM groups. However, mean white adipocyte area in visceral fat tissues was significantly smaller in the NFD group than in the HFD/PM group or HFD group, but white adipocyte area was significantly smaller in the HFD/PM group than in the HFD group (Fig. 1F and G). White adipocyte area to total adipocyte area ratios were calculated rather than measuring white adipocyte area, because white adipocytes were not found in perivascular fat in the NFD group. This ratio was significantly higher for perivascular fat in the HFD group than in the HFD/PM group (Fig. 1F and H, ***P=.001, vs NFD; $ $, P=.004, vs HFD). In subcutaneous fat tissues, the mRNA levels of adipogenesis factor such as Pparγ, Cebpa and Srebps [16,17] were higher in the HFD and HFD/PM groups than in the NFD group, and those levels were similar in

Serum levels of RAGE ligands, that is, AGE, HMGB1, and S100 β were significantly higher in the HFD groups than in the NFD group, but no difference was observed between the HFD and HFD/PM groups (Fig. 2E, G, and H). Serum AGE-albumin levels were significantly lower in the HFD/PM group than in the HFD group, but significantly higher in the HFD/PM group than in the NFD group (Fig. 2F). 3.5. Pyridoxamine affected RAGE accumulation and RAGE-RAGE ligand binding in fat tissues RAGE was located in the cell membrane of adipocytes. The expression level of RAGE in the subcutaneous, visceral, and perivascular adipocytes were higher in the HFD group, however, expression level of RAGE was similar in the HFD and HFD/PM groups in the subcutaneous adipocytes (Fig. 3A). RAGE accumulated significantly more in subcutaneous fat of the HFD and HFD/PM groups than of the NFD group; levels in the HFD and HFD/PM groups were not significantly different. RAGE accumulations were significantly lower in visceral and perivascular fat of the HFD/PM group than of the HFD group, but significantly higher in the HFD/PM group than in the NFD group (Fig. 3B, ***P=.000, vs NFD; $$ or $$$, P=.006 or P=.000, vs HFD). In subcutaneous fat, RAGE-AGE, RAGE-HMGB1, and RAGE-S100β binding were significantly higher in the HFD and HFD/PM groups than in the NFD group, but similar in the HFD and HFD/PM groups. However, in visceral and perivascular fat, RAGE-AGE, RAGE-HMGB1, and RAGE-S100β binding levels were significantly lower in the HFD/ PM group than in the HFD group, and levels in the HFD/PM group were significantly higher than those in the NFD group (Fig. 3C-E, ***P=.000, vs NFD; $$$, P=.000, vs HFD). 3.6. Pyridoxamine affected Glo-1 expression in fat tissues In subcutaneous fat, Glo-1 [21–23] mRNA expression was significantly lower in the HFD group than in the NFD or HFD/PM groups (Fig. 3F) whereas in visceral fat, Glo-1 mRNA expression

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Fig. 2. Accumulation of RAGE ligands in the fat tissues in HFD/PM treated rats(A-D) AGE (A), AGE-albumin (B), HMGB1 (C), and S100β (D) protein accumulations in subcutaneous, visceral, and perivascular fat tissues were greater in the HFD and HFD/PM groups than in the NFD group. However, no difference was observed between AGE, AGE-albumin, HMGB1 and S100β accumulations in the HFD and HFD/PM groups in subcutaneous fat and the levels of these ligands in the HFD/PM group were significantly lower than in the HFD group in visceral and perivascular fat. (E-H) Serum levels of AGE (E), HMGB1 (G) and S100β (H) were higher in the HFD group than in the NFD group, but serum levels in the HFD and HFD/PM groups were similar. (F) Serum levels of AGE-albumin were lower in the HFD/PM group than in the HFD group, but levels in the HFD/PM group were higher than in the NFD group. NFD: normal fat diet, HFD: high fat diet, HFD/PM: high fat diet with pyridoxamine, AGE: advanced glycation end-products, Alb: albumin, HMGB1: high motility group box 1 protein, S100β: S100 calcium-binding protein beta, SQ: subcutaneous fat, VIS: visceral fat, PV: perivascular fat. Results are presented as means±SDs. ***, significantly different (Pb.01) from the NFD group and $$, $$$, significantly different (Pb.01, Pb.001) from the HFD group.

was significantly lower in the HFD group than in the NFD and HFD/PM groups, and significantly higher in the HFD/PM group than in the NFD and HFD groups. In perivascular fat, Glo-1 mRNA expression was significantly lower in the HFD group than in the HFD/PM group, and significantly lower in the HFD/PM group than in the NFD group (Fig. 3F, *** or ***P=.04, **P=.03 or P=.008, vs NFD; $$, P=.001, vs HFD). Those patterns are same in the results which evaluated by western analysis (Fig. 3G). and immunofluorescence (Fig. 3H).

3.7. Pyridoxamine affected M1/M2 polarization in the fat tissues In subcutaneous fat tissues, the expression levels of Cd86 (M1 macrophage marker) [24,25] mRNA were not different in the three groups (Fig. 4A, * or **P=.03 or P=.006, vs NFD; $ or $$, P=.02 or P= .008, vs HFD). Also, the distribution level of the CD86 which evaluated immunofluorescence stain showed there was no difference between three groups (Fig. 4B). However, in visceral fat tissue, the CD86 mRNA expression levels were significantly lower in the NFD group than in the

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Fig. 3. Accumulations of RAGE and RAGE-RAGE ligand binding in the fat tissues of pyridoxamine treated rats on a high fat diet. (A) Expression level of RAGE of subcutaneous, visceral, and perivascular adipocytes were investigated by immunohistochemistry. (Original magnification 100× in subcutaneous fat and visceral fat and 200× in perivascular fat) (B) RAGE accumulations of subcutaneous, visceral, and perivascular fat were greater in the HFD and HFD/PM groups than in the NFD group, but in visceral and perivascular fat, RAGE accumulations were lower in the HFD/PM group than in the HFD groups. However, RAGE accumulations of subcutaneous fat were similar in the HFD and HFD/PM groups. (C-E) RAGEAGE (C), RAGE-HMGB1 (D), and RAGE-S100β (E) binding levels in all fat tissues sample in the HFD and HFD/PM groups were higher than in the NFD group. For in visceral and perivascular fat tissues levels of RAGE, RAGE-AGE, RAGE-HMGB1, and RAGE-S100β were lower in the HFD/PM group than in the HFD group, and these levels were higher in the HFD/PM group than in the NFD group except for subcutaneous fat tissues. (F) Regardless of fat tissue type, Glo-1 mRNA levels were lower in the HFD group than in the NFD or HFD/PM groups, but in subcutaneous fat Glo-1 mRNA levels were similar in the HFD and HFD/PM groups. In the perivascular fat, Glo-1 mRNA levels were higher in the HFD/PM group than in the HFD group, but lower in the HFD/PM group than in the NFD group. (G, H) Expression level of Glo-1 of subcutaneous, visceral, and perivascular adipocytes were investigated by immunoblotting and immunofluorescence.NFD: normal fat diet, HFD: high fat diet, HFD/PM: high fat diet with pyridoxamine, RAGE: receptor of advanced glycation end-products, AGE: advanced glycation end-products, HMGB1: high motility group box 1 protein, S100β: S100 calcium-binding protein beta, glo-1: glyoxalase I, SQ: subcutaneous fat, VIS: visceral fat, PV: perivascular fat. Scale bar=100 μm. Results are presented as means±SDs. *, ** and ***, significantly different (Pb.05, Pb.01 and Pb.001) from the NFD group and $$, $$$, significantly different (Pb.01, Pb.001) from the HFD group.

HFD or HFD/PM groups; levels were not significantly different in the HFD and HFD/PM groups. In perivascular fat tissue, the CD86 mRNA expression levels were significantly lower in the NFD group than in the HFD or HFD/PM groups, and significantly higher in the HFD group than in the HFD/PM group (Fig. 4A). Those patterns are same in the results which evaluated by immunofluorescence (Fig. 4B). In subcutaneous fat tissue, the mRNA expression level of Cd206 mRNA (M2 macrophage marker) [24,25] was similar in the three groups. However, the expression of Cd206 mRNA was significantly higher in the HFD/PM group than in the HFD group and its expression in the HFD/PM group was significantly lower than in the NFD group in visceral and perivascular fat (Fig. 4C, **P=.03, vs NFD; $, P=.03, vs HFD). The expression level of CD206 protein [24,25] was similar in the three groups showed same pattern by immunofluorescence (Fig. 4D). In visceral and perivascular fat, the expression of Cd206 RNA was significantly higher in the HFD/PM group than in the HFD group and its expression in the HFD/ PM group was significantly lower than in the NFD group.

HFD/PM groups than in the NFD group, but no significant difference was observed between the HFD and HFD/PM groups (Fig. 5A, C and E). However, in visceral and perivascular fat the mRNA expression levels of NF-κB, TNF-α, and IL-1β were significantly lower in the HFD/PM group than in the HFD group, and significantly higher in the HFD/PM group than in the NFD group (Fig. 5A, C and E). The translocation level of NF-κB and distribution expression level of, TNF-α, and IL-1β by the immunofluorescence stain showed significantly higher in the HFD and HFD/PM groups than in the NFD group, but no significant difference was observed between the HFD and HFD/PM groups in the subcutaneous fat tissue. However, in visceral and perivascular fat, the levels of NF-κB, TNF-α, and IL-1β were significantly lower in the HFD/PM group than in the HFD group, and significantly higher in the HFD/PM group than in the NFD group (Fig. 5B, D and F). 3.9. Pyridoxamine affected RAGE ligand production in AGE treated macrophages but not in AGE treated adipocytes

3.8. Pyridoxamine affected inflammation in fat tissues In subcutaneous fat, the mRNA expression levels of NF-κB [26], TNF-α [27–29], and IL-1β [30] were significantly higher in the HFD and

PM is known to reduce AGE formation. In the present study, PM was found to affect the accumulations and RAGE binding of RAGE ligands other than AGE. To determine how PM decreased HMGB1 and

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Fig. 4. M1/M2 polarization in the fat tissues of HFD/PM treated rats(A) In subcutaneous fat tissues, Cd86 mRNA levels were similar in the HFD, HFD/PM and NFD study groups. However, levels in visceral and perivascular fat tissues were lower in the NFD group than in the HFD and HFD/PM groups. In visceral fat tissues, Cd86 mRNA levels were similar in the HFD and HFD/ PM groups, but in the perivascular fat tissues, levels were higher in the HFD group than in the HFD/PM group. In the subcutaneous fat tissues. (C) Cd206 mRNA levels were similar in the three study groups, but in visceral and perivascular fat tissues, Cd206 mRNA levels were higher in the HFD/PM group than in the HFD group, but lower in the HFD/PM group than in the NFD group. (B, D) Expression level of CD86, CD206 of subcutaneous, visceral, and perivascular adipocytes were investigated by immunofluorescence. NFD: normal fat diet, HFD: high fat diet, HFD/PM: high fat diet with pyridoxamine, SQ: subcutaneous fat, VIS: visceral fat, PV: perivascular fat. Scale bar=50 μm. Results are presented as means±SDs. Scale bar=100 μm *, **, significantly different (Pb.05, Pb.01) from the NFD group and $, significantly different (Pb.05) from the HFD group.

S100β levels in fat tissues, macrophages and adipocytes were treated with AGE without PM or with AGE and PM and levels of HMGB1 and S100β were compared with those in non-treated controls.

In macrophages, HMGB1 and S100β levels were significantly higher after AGE/PM treatment than after no treatment, but significantly lower than after AGE treatment (Fig. 6A and B). However,

Fig. 5. Expressions of genes related to inflammation in the fat tissues of pyridoxamine treated rats on a high fat diet. (A, C, E) In subcutaneous fat tissues, the mRNA levels of NF-κB (D), TNF-α (E), and IL-1β (F) were higher in the HFD and HFD/PM groups than in the NFD group, and levels were similar in the HFD and HFD/PM groups. However, in visceral and perivascular fat tissues the mRNA levels of NF-κB, TNF-α, and IL-1β were lower in the HFD/PM group than in the HFD group, and levels in the HFD/PM group were higher than in the NFD group. (B, D, F) Expression level of NF-κB, TNF-α and IL-1β of subcutaneous, visceral, and perivascular adipocytes were investigated by immunofluorescence. NFD: normal fat diet, HFD: high fat diet, HFD/PM: high fat diet with pyridoxamine, NF-κB: nuclear factor-kappa Β, TNF-α: tumor necrosis factor-alpha, IL-1β: interleukin-1 beta, SQ: subcutaneous fat, VIS: visceral fat, PV: perivascular fat. Results are presented as means±SDs. Scale bar=100 μm **, ***, significantly different (Pb.01, Pb.001) from the NFD group and $$, $$$, significantly different (Pb.01, Pb.001) from the HFD group.

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Fig. 6. RAGE ligand production in AGE treated macrophages and adipocytes(A, B) In the AGE treated macrophages, the protein levels of HMGB1 and S100β were higher cells treated with PM plus AGE than in non-treated cells, but were lower in PM plus AGE treated cells than in AGE treated cells. (C, D) In AGE treated adipocytes, the protein levels of HMGB1 and S100β were higher in PM plus AGE treated cells than in non-treated cells, but were similar in PM plus AGE and AGE treated group. PBS: PBS treated group, AGE: AGE treated group, AGE/PM: AGE with pyridoxamine treated group. AGE: advanced glycation end-products, HMGB1: high motility group box 1 protein, S100β: S100 calcium-binding protein beta, PM: pyridoxamine. Results are presented as means±SDs. ***, significantly different (Pb.001) from non-treated controls and $$, significantly different (Pb.01) from AGE treated cells.

for adipocytes, HMGB1 and S100β levels were significantly higher after AGE/PM treatment than after no treatment, and non-significantly different after AGE/PM or AGE treatment (Fig. 6C and D).

4. Discussion The present study shows (1) pyridoxamine attenuated HFD induced weight gain (2) pyridoxamine attenuated adipocyte hypertrophy in the visceral fat tissue, not in the subcutaneous tissue (3) pyridoxamine increase lipolysis and decrease adipogenesis in the visceral and perivascular fat tissue but not in the subcutaneous tissue (4) pyridoxamine attenuated RAGE ligands (AGE, AGE-albumin, HMGB1, and S100β) accumulation in the visceral and perivascular fat tissue but not in the subcutaneous tissue (5) RAGE-RAGE ligands binding amount in the fat tissue is attenuated by pyridoxamine in the visceral and perivascular fat tissue, not in the subcutaneous tissue (6) pyridoxamine attenuated M1 increasing and M2 decreasing in the visceral and perivascular fat tissue, not in the subcutaneous tissue (7) pyridoxamine induced Glo-1 expression in the visceral fat of HFD, even more than normal fat diet, while Glo-1 expression of perivarscular fat tissue induced by pyridoxamine was not higher than normal fat diet (8) Pyridoxamine attenuated inflammatory factors such as NFκB, TNF-α, and IL-6 level in the visceral and perivascular fat tissue, not in the subcutaneous tissue (9) pyridoxamine attenuated release of RAGE ligands from AGE treated macrophage, however those attenuation effect was not significant in the AGE treated adipocyte. Adipocyte size influences the expressions of inflammatory mediators, and adipocyte hypertrophy promotes the productions of IL-6, IL8, Mcp-1 and granulocyte colony-stimulating factor [31–33]. It has been previously reported pyridoxamine attenuated weight gain and adipocyte size increases in the visceral fat tissue of HFD fed mice [12]. The present study shows the suppression of adipocyte size increases

by pyridoxamine depends on fat tissue type, and that this effect is marked in visceral fat but not in subcutaneous fat. There are two types of perivascular fat, that is, brown and white perivascular fat. Brown perivascular fat is associated with the regulation of vascular function and with intravascular temperature regulation [34–36], whereas white perivascular fat has features similar to white adipose tissue, which induces inflammation and is highly responsive to HFD [37]. In the present study, no white perivascular adipocytes were observed in the NFD group, but they were observed in the HFD group, and white perivascular adipocyte counts were lower in the HFD/PM group than in the HFD group. Our study results showed that pyridoxamine attenuated the increase of mRNA levels which related with adipogenesis induced by HFD in the visceral and perivascular fat tissue not in the subcutaneous fat tissue. Pyridoxamine induced lipolysis in the visceral and perivascular fat tissue not in the subcutaneous fat tissue of HFD fed animals. RAGE ligands, such as, AGEs, HMGB, and S100β, accumulate in visceral fat in animals fed a HFD [8,9]. Similarly, we observed accumulations of AGE, AGE-albumin, HMGB, and S100β in the visceral fat of rats fed a HFD. Previous studies have usually reported accumulations of RAGE ligands in visceral fat tissues. In the present study, we examined RAGE ligand accumulations in different fat tissues in rats fed a HFD, and found they accumulated in subcutaneous and perivascular fats. Interestingly, we also observed pyridoxamine suppressed accumulations in visceral and perivascular fat, but not in subcutaneous fat, and that these attenuations were far more prominent in tissues than in serum. In fat tissues, RAGE-RAGE ligand binding exhibited patterns similar to those of RAGE ligand accumulations. Pyridoxamine decreased RAGE-RAGE ligand binding levels in visceral and perivascular fats of HFD fed rats, but no prominent reduction in binding was observed in subcutaneous fat.

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In addition, we evaluated M1/M2 polarization, which is known to be related to inflammatory status [38]. Moreover, AGE is known to induce M1 polarization via a RAGE-dependent process [11]. M1 polarization was found to be elevated in visceral and perivascular fat in the HFD group, but not in subcutaneous fat, whereas M2 polarization decreased in visceral and perivascular fat in the HFD group, but not in the subcutaneous fat. M1 and M2 phenotypes are defined by microenvironment, for example, M1 macrophages are generated by treating bone-marrow-derived macrophages with lipopolysaccharide and interferon-γ or other pro-inflammatory cytokines, while M2 macrophages are generated by treating then with IL-4, IL-13 or IL-10 [38]. Accordingly, M1 macrophages are considered to be proinflammatory, and notably express TNF-α and IL-1β [38]. Most of macrophages at sites of inflammation attributed to inflammatory diseases are considered to be M1 macrophages [38]. By contrast, M2 macrophages are thought to play more diverse roles, in anti-inflammatory pathways, tissue remodeling, and wound healing [38]. It has been established obesity induces exclusive the polarization of macrophages in fat tissues from the M2 to the M1 phenotype [38]. In the present study, RAGE ligand accumulation and M1 polarization differed in subcutaneous, visceral, and perivascular tissues, which may explain why perivascular and visceral fat tissues induce more inflammation than subcutaneous fat tissues. In the present study, pyridoxamine decreased M1 polarization after HFD in visceral and perivascular fats, but not in subcutaneous fat. However, this effect of pyridoxamine in visceral fat was not significant, but rather showed a tendency to decrease. Nevertheless, this effect of pyridoxamine was prominent in perivascular fat. In addition, pyridoxamine caused an increase in M2 polarization after HFD in visceral and perivascular fat, but not in subcutaneous fat. Furthermore, pyridoxamine attenuated the expressions of the inflammatory cytokines NF-κB, TNF-α, and IL-1β in visceral and perivascular fats, but not in subcutaneous fat. Several authors have suggested that M2 polarized macrophages in adipose tissue are associated with catabolism and energy expenditure [8,21], and in one study, the increase in metabolic rate induced by M2 polarization in RAGE null mice was found to have a protective effect on HFD-induced obesity [8]. The glyoxalase Glo-1 importantly protects against AGE formation [21]. In animal models of obesity, Glo-1 levels have been reported to be decreased in liver [39], in glomeruli [40] and in neuronal tissues [41], and it is known Glo activity is downregulated by AGE-RAGE [42] or inflammation [43]. Furthermore, in a previous study, Glo activity was diminished by HFD and by pyridoxamine-induced Glo-1 activity in visceral fat tissues [12]. In the present study, Glo-1 expression was lower in subcutaneous, visceral, and perivascular fat tissues in the HFD group than in the NFD group, and pyridoxamine increased Glo-1 expression in visceral and perivascular fat tissues. In the present study, pyridoxamine inhibited HFD-induced AGE formation and reduced the HFD-induced up-regulations of other RAGE ligands, such as, HMGB1 and S100β. When we treated RAW 264.7 macrophages with AGE, they released HMGB 1 and S100β and this release was suppressed when AGE pretreated macrophages were treated with pyridoxamine. On the other hand, when we treated adipocytes with AGE, the releases of HMGB1 and S100β were increased, but when AGE pretreated adipocytes were treated with pyridoxamine, these increases were not diminished. Taken together, we suggest AGE induces the release of other RAGE ligands (HMGB1 or S100β) from macrophages, and that when these ligands bind with RAGE they induced an increase in M1 and a decrease in M2 polarization and create an inflammatory environment. In addition, RAGE/RAGE ligand binding decreased Glo-1 activity, increased AGE accumulation in fat, and further induced RAGE ligand release by macrophages. It would appear that pyridoxamine might block RAGE ligand production by macrophage, and thus attenuate RAGE ligand accumulation, reduce RAGE/RAGE ligand binding, reduce

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M1 polarization, and increase Glo-1 levels in fat tissues. Furthermore, we found these effects of pyridoxamine differed in subcutaneous, visceral, and perivascular fat tissues. In particular, pyridoxamine attenuated M1 polarization and increased Glo-1 expression in perivascular and visceral fat tissues, which are main fat tissues associated with inflammation. These findings suggest pyridoxamine be considered a candidate treatment for obesity or complications related to obesity-associated inflammation. Previous animal studies that evaluated the effect of pyridoxamine on diabetic kidney disease using a rat administered 1-2 g of pyridoxamine in 1 L of drinking water [33,34], and thus, pyridoxamine doses were uncertain. Previous studies showed various range of actual pyridoxamine dose (from 60 mg/kg per day to 200 mg/kg per day) which animal was taken, even though animal allowed to drink pyridoxamine water of 1–2 g/L [44,45]. In the present study, a fixed dose of pyridoxamine (2 mg/day) was administered to each animal based on dosages used in human clinical trials on the use of pyridoxamine for the treatment of diabetic kidney disease. In these clinical trials the dosages of pyridoxamine used were 100, 300, and 600 mg/day [14]. Even about 5.71 mg/kg per day of pyridoxamine which is quite lower dose than other animal studies had used attenuated the weight increase and M1 polarization which related with inflammation of fat tissue in our study. Limitation of our study is that we cannot be sure RAGE pathway really do main role on changes of M1/M2 polarization, inflammatory cytokine level, and Glo-1 expression and RAGE pathway induce those changes differently depends on the fat tissue location in the HFD fed animal after pyridoxamine administration, because we did not evaluate the pyridoxamine effect in the RAGE null mice or animal which are administered the RAGE blocker. However, we can assume that RAGE pathway do main role on the attenuation effect by pyridoxamine on adipocytes hypertrophy, because the RAGE pathway is a common pathway of M1/M2 polarization, inflammatory cytokine level increase, and Glo-1 expression changes. In conclusion, the present study shows pyridoxamine suppressed HFD-induced weight increases and M1 polarization, and increased Glo-1 expression through a RAGE pathway in the perivascular and visceral fat tissues of HFD-induced obese rats and RAGE pathway might induce those attenuation effects of pyridoxamine. These findings suggest pyridoxamine is a candidate treatment for obesity or complications related to obesity associated inflammation. Acknowledgements This research was supported by the Basic Science Research Program through the National Research Foundation of Korea [grant number, 2016R1D1A1B03933680, KH]; and the Jeisys of Korea [grant number, 20175274, KB]. Disclosure/conflict of interest The authors declare that they have no conflict of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jnutbio.2019.04.001. References [1] Ibrahim MM. Subcutaneous and visceral adipose tissue: structural and functional differences. Obes Rev 2010;11(1):11–8. [2] Bruun JM, Lihn AS, Pedersen SB, Richelsen B. Monocyte chemoattractant protein-1 release is higher in visceral than subcutaneous human adipose tissue (AT): implication of macrophages resident in the AT. J Clin Endocrinol Metab 2005;90 (4):2282–9.

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