3T3-L1 Adipocyte Culture
Cells were obtained from ATCC (Manassas, VA) and cultured according to standard conditions. Briefly, cells were grown under 5% CO2 in Dulbecco's Modified Eagles Medium (DMEM) containing 10% fetal bovine serum (Hyclone, Logan, UT) and 0.5% penicillin-streptomycin mixture (Invitrogen, Carlsbad, CA). Cells were allowed to reach confluence, and two days post confluence (day 0), were induced to differentiate with a medium containing 10% fetal bovine serum, 1.7 μM insulin, 1 μM dexamethasone, and 0.5 mM IBMX for 48 h. Thereafter, fresh medium containing only insulin and fetal bovine serum was added for another 2 days. From then on media was replenished every 2 days with DMEM containing only 10% FBS. Fully differentiated cells were treated for 24 hours with either Staphylococcus aureus derived peptidoglycan (10 μg/mL) or E.coli lipopolysaccharride (100 ng/mL) (Sigma, St. Louis. MO).
Eight week old male C57BL/6J mice were fed either a high fat diet (HF, D12492i) with 60% fat calories (n = 8) or a control diet (LF, D12450Bi) with 10% calories (n = 8) from fat (Research Diets, New Brunswick, NJ, http://www.researchdiets.com) for 12 weeks. At the end of the experiment animals were euthanized by CO2 asphyxiation followed by cervical dislocation. All animal care protocols were approved by the Purdue Animal Care and Use Committee. Epididymal adipose tissue was obtained by careful dissection of adipose tissue around the epididymis and used for RNA extraction with Trizol (Invitrogen, Carlsbad, CA) or tissue lysates for western blotting. We also collected subcutaneous (collected from underneath the skin around the lumbar area), mesenteric (collected by careful dissection of adipose tissue from around the intestine) for a comparative analysis of SLPI mRNA expression by real-time PCR. To determine the relative expression of SLPI in adipocytes and stromal vascular fraction (SVF), adipose tissue was subjected to collagenase digestion (1 mg/ml Collagenase type 1, Sigma) in Krebs Ringer Buffer (118.5 mM NaCl, 4.8 mM KCl, 2.7 mM CaCl2, 1.2 mM KH2PO4, 1.1 mM MgSO4, 7H2O, 25 mM NaHCO3, 5 mM glucose and 5% (w/v) BSA, pH 7.4) with shaking at 150 RPM for 30 minutes at 37°C. After digestion, adipocytes were allowed to separate by flotation and the infranatant solution was centrifuged for 5 minutes at 300 g to pellet the stromovascular fraction (SVF). The adipocyte fraction was washed three times with the KRB buffer to remove contaminants and ensure a pure population of adipocytes. This method has been validated with flow cytometry to yield a 100% pure population of adipocytes. Subsequently, RNA was isolated from adipocytes and the SVF for comparison with whole adipose tissue.
Anti-inflammatory effect of SLPI
Differentiated 3T3-L1 adipocytes were pretreated for 2 hours with 10 ng/ml recombinant human SLPI (R &D Systems, Minneapolis, MN) and then treated with LPS for 3 hours. Media was recovered for ELISA and RNA for RT-PCR.
Real-time quantitative RT-PCR
Total RNA from treated cells was extracted with Trizol Reagent (Invitrogen) according to the manufacturer's protocol. The mRNAs were treated with Turbo DNase (Ambion, Austin, TX) to remove contaminating DNA and reverse transcribed into cDNA using Improm II reverse transcriptase (Promega, Madison, WI). Real-time PCR was performed using a MyIQ real-time PCR detection machine (Bio-Rad) with the Faststart SYBR green based mix (Roche, Indianapolis, IN). Primers sequences used were: IL-6, 5'-AACGATGATGCACTTGCAGA-3' and 5'-GAGCATTGGAAATTGGGGTA-3' for the sense and antisense primers, respectively (14); SLPI, sense, 5'-TGCTTAACCCTCCCAATGTC-3' and antisense, 5'-AATGCTGAGCCAAAAGGAGA-3'; β-actin sense, 5'-ATGGGTCAGAAGGACTCCTACG-3' and antisense, 5'-AGTGGTACGACCAGAGGCATAC-3'; TNFα, 5'-AGCCCCCAGTCTGTATCCTT-3' and 5'-CTCCCTTTGCAGAACTCAGG-3'. Quantification of transcripts was done with the ΔΔ Ct method with normalization against the β-actin.
Whole tissue lysates were obtained by homogenizing tissues and cells in RIPA lysis buffer (0.5 M Tris-HCl, 1.5 M HCl, 2.5% Deoxycholic acid, 10% NP-40 and 10 mM EDTA) supplemented with protease and phosphates inhibitor cocktail (Sigma). Homogenized tissues and cells were then cleared of cellular and tissue debris by centrifugation at 10,000 g for 10 minutes at 4°C. Protein concentrations were determined with the BCA kit (Sigma). For immunoblotting, 50 μg of lysates were resolved on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. Membranes were probed with rabbit anti-SLPI (Cat # SC-28803, Santa Cruz, CA, USA) primary antibody and HRP-conjugated anti-rabbit secondary antibody (Cat# 7074, Cell Signaling, Danvers, MA, USA). To determine the role of IKBα protein in the regulation of SLPI effect, the expression of phosphorylated and native IKBα was quantified by western blotting using rabbit primary antibodies (Cat# 2859 and 4812, Cell Signaling, Danvers, MA, USA). Blots were subsequently blotted with the Supersignal® West Pico chemilumniscent reagent (Pierce, Rockford, IL) and exposed to autoradiographic film to capture protein specific signals.
ELISA for Media IL-6
Media concentration of IL-6 was determined with a mouse IL-6 ELISA kit (Endogen, Rockford, IL) according to the manufacturer's instructions. This kit has an assay sensitivity of < 7 pg/ml and an inter assay and intra assay variation of < 10%.
All data were checked for normality and then analyzed using the GLM model analysis. When treatment effects were significant, mean separation was accomplished using the least-squares mean separation procedure.