Glut4

The protein expression in NRVM or rat heart were analyzed by western blotting with method described previously [26 (link)]. Briefly, equal amounts of protein samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Perkin-Elmer Life Sciences, Boston, MA, USA). The membranes were blocked in 5% fat-free milk dissolved in TBST (Tris/phosphate/saline/Tween) and incubated with primary antibody to p-AMPK, p-AKT, caspase-3 (Cell Signalling, Beverly, MA, USA), GLUT4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and β-actin (Sigma-Aldrich).

Proteins were extracted from the heart tissue in a 1x RIPA buffer (Melford, Ipswich, UK). The protein concentration was determined with Bradford assay. Total protein homogenates 20 μg/30 μl were denatured, separated on sodium dodecyl sulfate polyacrylamide electrophoresis gels, and transferred to a polyvinylidene fluoride membrane (Millipore, Darmstadt, Germany). The membrane was blocked with 2.5 % BSA in Tris- Buffered Saline Tween 20 for 1 h, before being incubated overnight at 4 °C with CD36 (1:1000, Cell Signalling Technology, Cambridge, UK), GLUT-4 (1:100, Santa Cruz, Biotechnology, Heidelberg, Germany), insulin receptor-β (1:1000, Cell Signalling Technology, Cambridge, UK), phosphorylated-PI3K (1:1000, Cell Signalling Technology, Cambridge, UK), PI3K (1:1000, Cell Signalling Technology, Cambridge, UK), AKT, or phosphorylated-AKT (1:500, Abcam, Cambridge, UK). After washing the blots to remove excessive primary antibody binding, the blots were incubated for 1 h with a horseradish peroxydase conjugated secondary antibody at room temperature. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), housekeeping protein, was used for loading control and protein normalization. The immunoreactive protein bands were developed using the Enhanced Chemiluminescence system (PerkinElmer, Rodgau-Juegesheim, Germany). The intensity of the immunoblot bands was detected with Chemismart 5100.

The 3T3-L1 cells were graciously provided by Dr. Jae-Woo Kim (Yonsei University, College of medicine). Oligonol was supplied by KCF Korea Co. (Seoul, Korea). The triglyceride quantification kit was purchased from BioVision (Mountain View, CA, USA). Antibodies for experiments are the following: CEBPα, pACC(Ser79), ACC, FAS, pAKT(Ser473), AKT, pFOXO1(Thr24), FOXO, pAMPK(Thr172), AMPK, pmTOR(Ser2448), mTOR, pS6K(Thr389), S6K, pS6 (Cell Signaling, Danvers, MA, USA), PPARγ, GLUT4, Adiponectin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), aP2 (Cayman, Ann Arbor, MI, USA), and β-actin (Sigma-Aldrich, Oakville, Canada).

Five-micrometer paraffin-embedded gonadal WAT (gWAT) and interscapular BAT (iBAT) sections were hydrated and blocked with 2.5% normal goat serum–PBST (phosphate-buffered saline, 0.1 % Tween-20) for 1 h at room temperature. Primary antibodies (GLUT4, 1:50, Santa Cruz; F4/80, 1:10, Invitrogen; UCP1, 1:500, custom made) were applied overnight at 4 °C. After washing three times with PBST, secondary antibody against rabbit (SignalStain Boost IHC, Cell Signaling) was applied for 1 h at room temperature and developed with the DAB Kit (Vector Laboratories) according to the manufacturer's instructions. WAT sections were counterstained with hematoxylin before dehydration. Standard hematoxylin and eosin (H&E) staining was performed with BAT sections. Sections were mounted with RotiHistokit (Carl Roth). Quantification of lipid droplet sizes in H&E-stained BAT sections was analyzed and calculated using the Adiposoft plugin for Fiji (ImageJ). Large lipid droplets were defined as surface >300 μm². One 20x magnification frame of BAT per mouse was scored.

Primary antibodies against Fascin-1 [mouse monoclonal (D-10) sc-46675], RhoA [mouse monoclonal (26C4) sc-418], GAPDH [rabbit polyclonal (FL-335) sc-25778], Actin [goat polyclonal (C-11) sc-1615], GLUT1 [mouse monoclonal (A-4) sc-377228], GLUT4 [rabbit polyclonal (H-61) sc-7938] and SDF-1/CXCL12 [rabbit polyclonal (FL-93) sc-28876] were from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA); anti-phospho-p44/42 ERK (Thr202/Tyr204) [mouse monoclonal, #9106S] and p44/42 ERK1/2 [rabbit polyclonal, #9102] from Cell Signaling Technology, Inc. (Danvers, MA, USA); anti-FAK [rabbit polyclonal, GTX132141] was from GeneTex, Inc. (Irvine, CA, USA); anti-MEK1[rabbit monoclonal, #04-376] was from EMD-Millipore (Burlington, MA, USA); anti-α-smooth muscle Actin (mouse monoclonal (1A4), #A2547) was from Sigma-Aldrich (Saint Louis, MO, USA). Rabbit (#A0545) and goat (#A4174) peroxidase-conjugated secondary antibodies, media and sera for cell cultures were purchased from Sigma-Aldrich; mouse peroxidase-conjugated secondary antibody (#sc-2005) was from Santa Cruz Biotechnology, Inc. Plasticware was obtained from Corning Inc. (Corning, NY, USA). 2-deoxy-[3H]-D-glucose was provided by Perkin Elmer (Waltham, MA, USA). Other reagents for cell culture and microscopy were obtained from Sigma-Aldrich, except where otherwise indicated.

For Western, the following antibodies were used: adipophilin (1:10,000, guinea pig) and perilipin (1:100,000, guinea pig) from Fitzgerald Industries International (Acton, MA, USA); CHOP (1:200, rabbit), SREBP1 (1:200, mouse), NFkB (1:200, mouse), PPARγ (1:200, rabbit), GLUT4 (1:200, mouse) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-KDEL (BiP, 1:2500, mouse) from MBL International (Woburn, MA, USA); anti-caspase 3 (1:1000, rabbit) from Cell Signaling Technology (Danvers, MA); FAS (1:3000, mouse) from BD Transduction Laboratories (Franklin Lakes, NJ, USA); actin (1:10,000, rabbit), and polyubiquitin (1:500, rabbit) from Sigma-Aldrich; Hsp70 (1:1000, mouse) from Stressgen Bioreagents (Victoria, BC, Canada), while anti-β1 (1:5000 rabbit) and anti-S4 (1:2000, rabbit) antibodies were custom obtained 24 (link). Secondary antimouse, anti-rabbit and anti-guinea pig Fab' fragments labelled with horseradish peroxidase (HRP) or alkaline phosphatase (AP) were from Jackson Immunoresearch (West Grove, PA, USA).

The myotubes were incubated for 20 min in DMEM-low glucose-Glutamax in the presence or absence of 100 nM insulin. Afterwards, the cells were harvested in a RIPA buffer (Sigma, St. Louis, MO, USA) complemented with 10 µL/mL protease inhibitor, 10 µL/mL phosphatase I inhibitor and 10 µL/mL phosphatase II inhibitor. Afterwards, the protein concentration was determined by BCA reagent (Thermo Scientific, Rockfold, IL, USA). 20 µg of total protein were run on 4–15% Mini-PROTEAN^® TGX™ Precast Gels (Bio-Rad, Hercules, CA, USA), electroblotted onto PVDF membranes (Millipore, Bradford, MA, USA), and immunodetected with ChemiDoc MP imaging system (BioRad, Hercules, CA, USA) while using the following primary antibodies: GLUT4 (Santa Cruz Biotech, CA, USA), Ser9 pGSK-3β, Thr172pAMPK, Ser473 pAkt (Cell Signaling Technology, Danvers, MA, USA), Tyr-989 pIRS-1 (Abcam, Cambridge, UK), and Thr642 pAS160 (Gene Tex, CA, USA). Histone H3 (Cell Signalling Technology, Danvers, MA, USA) served as an internal control, with the exception of pAkt, where the internal control was GAPDH (Cell Signalling Technology, Danvers, MA, USA). The bound antibodies were visualized by an ECL system (Thermo Fisher Scientific Inc., Rockford, IL, USA) and quantified using Chemi-Doc MP imaging system (Bio-Rad, Hercules, CA, USA).