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

 Pyridoxamine reduced HFD-induced weight gain, attenuated adipocyte size increases, RAGE ligand accumulations, and RAGE-RAGE ligands binding, and decreased macrophage M1 polarization and increased M2 polarization in the visceral, subcutaneous, and perivascular fat tissues of Sprague-Dawley rats fed a high fat diet (HFD)

• These findings suggest pyridoxamide is a candidate for the treatment of obesity or complications related to obesity-induced inflammation.

Receptor for advanced glycation end products (RAGE)

• In the obese, visceral fat tissue produces more inflammatory cytokines and is more infiltrated by inflammatory cells (predominantly macrophages) than subcutaneous fat tissues
• This tissue type is particularly susceptible to inflammation, as evidenced by the fact that levels of monocyte chemoattractant protein (MCP-1) are more than 40-fold higher in perivascular adipocytes than subcircaneous adipocytes
• RAGE activates inflammation-related signaling cascades involving nuclear factor-(NF)κB, ERK (extracellular signal-regulated kinase) 1/2, p38 MAPK, JNK (c-Jun N terminal kinases), PKC (protein kinase C), Rac/Cdc42, or TIRAP and MyD88
• Several studies have shown RAGE is related to obesity, 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

Animals

• 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.
• 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 2 mg/day of orally administered daily for another 4 weeks with the same diet. After these periods, animals were sacrificed

Cell culture

• 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 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, 1 μM dexamethasone, and 0.5 mM methylisobutylxanthine for 9 days.

Sample preparation

• 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 overnight at 4°C and then placed in an automatic dehydration machine (ASP300S, Leica, Milton Keynes, UK).
• Absolute concentrations of RAGE and RAGE were determined using ELISA (Myriad, USA; Mycam, Cambridge, UK) and quantified using qRT-PCR (quantitative real-time polymerase chain reaction), cDNA was synthesized from 1 μg of total RNA using a PrimeScript 1st strand cDNA Synthesis Kit (#6110A, TAKARA, Shiga, Japan), and for preparation serum, collected blood was incubated at room temperature for 20 minutes, then centrifuged at 3,000 rpm for 10 minutes and aliquoted supernatant.

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
• 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 in blocking solution overnight at 4°C, washed with TBST, incubate with appropriate secondary antibodies, and rewashed.

Immunofluorescence (IF)

• The fat paraffin tissue slides were removed, incubated in normal animal serum to block antibody binding, and then rinsed twice with PBS.
• Fluorescence was detected by a confocal microscope using qRT-PCR to determine levels of Cd86 and Cd206, Glo-1 and NF-κB, TNF-α and IL-1β, inflammation factors.

Results

• Animals in the HFD group gained weight significantly more than animals in the NFD group (Fig. 1A).
• The relationship between body weight, triglyceride level, and adipocyte size in the high fat diet and pyridoxamine treated rats was similar.
• Mean triglyceride, total cholesterol, glucose, and serum triglyceride levels were all significantly lower in HFD/PM group than in the NFD group, and no difference was observed between the two groups after 2 months of normal diet and PM/HFD.

Discussion

• The present study shows that pyridoxamine attenuated HFD induced weight gain, adipocyte hypertrophy in the visceral fat tissue, not in the subcutaneous tissue, and increased adipocyte lipolysis and decrease adipogenesis in the perivascular fat tissue.
• Pyridoxinamine suppressed accumulations of RAGE ligands (AGE, AGE-albumin, HMGB1, and S100β) in visceral fat, but not in subcutaneously fat tissue (5).
• RAGE-RAGE ligands binding amount in fat tissue is attenuated by Pyridoxideamine in the abdominal fat tissue of HFD-fed rats, whereas it is not found in fat-free tissue of non-fed animals (6)
• Adipocyte size influences the expressions of inflammatory mediators (i.e., IL-6, IL-8, Mcp-1 and granulocyte colony-stimulating factor) in rats fed a HFD, and suppression of adipocyte size increases by pyridooxamine depends on fat tissue type, and this effect is marked in visceral Fat tissue type.

Reference

• 10.1016/j.jnutbio.2019.04.001