All chemicals were obtained from Sigma-Aldrich unless otherwise stated. IKKε (2690), TBK1 (3013), phosphorylated TBK1 (Ser172-5483), IKKα/β (2684), phosphorylated IKKα/β (ser176-2687) AKT (9272), phosphorylated AKT (Ser473-9272), S6K(2708 diluted 1:250), phosphorylated S6K (Thr389-9205), S6 (2317), phosphorylated S6 (Ser235/236-2211), IRF3 (4302), phosphorylated IRF3 (Ser396-4947), HSL (4107), phosphorylated HSL (Ser563-4139 or Ser660-4126), and PPARγ (2443)-specific antibodies were purchased from Cell Signaling. RalA (610222)-specific antibody was obtained from BD Bioscience. UCP1 (UCP11-A-diluted 1:250 for WAT detection)-specific antibody was obtained from Alpha Diagnostics. All antibodies were diluted 1:1,000 unless otherwise noted. Enhanced chemiluminescence reagents were purchased from NEN, Inc. EDTA-free protease inhibitor tablet was purchased from Roche Diagnostics.
Animals and animal care
We fed wildtype male C57BL/6 mice a HFD consisting of 45% of calories from fat (D12451 Research Diets Inc.) starting at eight weeks of age for 12–24 weeks, while ND C57BL/6 controls were maintained on normal chow diet consisting of 4.5% fat (5002 Lab Diet). We fed C57BL/6 diets containing ω-3 fatty acids as previously described 25
. Rosiglitazone treatment was administered for three weeks by addition of the compound to the diet in mice that had been on HFD for 16 weeks. Each mouse consumed on average 3.5 mg per kg rosiglitazone per day. Amlexanox was administered by daily oral gavage. For the prevention groups, amlexanox (25 mg per kg or 100 mg per kg) administration was begun concurrently with HFD feeding at eight weeks of age. For the treatment groups, 25 mg per kg amlexanox treatment was begun at 20 weeks of age after 12 weeks of HFD. To test the effect of amlexanox withdrawal, mice in the treatment group were switched from amlexanox gavage to vehicle control after eight weeks of amlexanox treatment. We fed control and ob/ob mice a normal chow diet and gavaged with 100 mg per kg amlexanox or vehicle control beginning at ten weeks of age. Animals were housed in a specific pathogen-free facility with a 12-hour light/12-hour dark cycle and given free access to food and water. All animal use was in compliance with the Institute of Laboratory Animal Research Guide for the Care and Use of Laboratory Animals and approved by the University Committee on Use and Care of Animals at the University of Michigan and UCSD.
The remaining weight of food provided was determined daily for singly housed mice. Daily food consumption was calculated from a three-day average.
Energy expenditure and respiratory quotient
C57Bl6 mice in the amlexanox treatment group were placed in metabolic cages. The University of Michigan Metabolic Phenotyping Core measured oxygen consumption (VO2
), carbon dioxide production (VCO2
) and spontaneous motor activity during 3 consecutive days using the Comprehensive Laboratory Monitoring System (Columbus Instruments), an integrated open-circuit calorimeter equipped with an optical beam activity monitoring system 53
. Values presented are normalized to body weight. The respiratory quotient was calculated by dividing carbon dioxide production by oxygen consumption. We used the mean values for light and dark cycles to analyze statistical significance.
The University of Michigan Metabolic Phenotyping Core used NMR analysis to quantify body fat, lean body mass and fluid content in ob/ob mice and C57BL/6 mice in the amlexanox treatment group.
Hyperinsulinemic Euglycemic clamp
C57Bl6 mice placed on HFD starting at eight weeks of age, starting at 16 weeks of age mice were gavaged daily with amlexanox or vehicle control. After four weeks of treatment, the right jugular vein and carotid artery were surgically catheterized and mice were given 5 days to recover from the surgery. During this recovery period, amlexanox was delivered in drinking water. On the day of the clamp a final oral gavage treatment was performed. After a 5–6 hour fast, hyperinsulinemic clamp studies were performed on conscious mice using the protocol adopted from the Vanderbilt Mouse Metabolic Phenotyping Center by the University of Michigan Animal Phenotyping Core consisting of a 90 min equilibration period, followed by a 120 min experimental period (t = 0 to 120 min). Insulin was infused at 4.0 mU kg−1 min−1. This experiment was performed at the University of Michigan Metabolic Phenotyping Core. We measured serum free fatty acid levels using the colorimetric NEFA-HR(2) kit (Wako).
Core body temperature
We performed rectal temperature measurements using a YSI 4600 Precision thermometer (YSI, Inc.).
Blood chemistry analysis
Blood glucose was measured by OneTouch Ultra Glucometer. Plasma from mice fasted for six hours was isolated from whole blood collected into heparinized tubes. We used an insulin ELISA kit (Crystal Chem Inc.) to measure serum insulin concentrations. Leptin and adiponectin levels were measured by ELISA kits purchased from Cayman Chem Inc. We quantified serum cytokine levels using luminex technology in a multi-analyte panel plate purchased from Millipore. Additionally, we measured TNF-α levels using ELISA kits purchased from R&D Systems.
Glucose and insulin tolerance tests
For glucose tolerance tests, after a six-hour fast, we gavaged mice with glucose at a dose of 1.5 g per kg (C57BL/6 mice) or 1.2 g per kg (ob/ob mice). For insulin tolerance tests, mice were fasted for three hours then given an intraperitoneal injection of insulin (1.2 units per kg for C57BL/6 mice and 2.0 units per kg for ob/ob mice). We measured blood glucose at basal, 15, 30, 45, 60, 90, 120 and 180 minutes from tail blood using the OneTouch Ultra glucometer (Lifescan).
Tissue lipid content
We isolated liver lipids by ethanolic KOH saponification and BAT lipids by chloroform extraction and then triglyceride levels were quantified using the Triglyceride Reagent kit (Thermo Scientific).
Liver glycogen content
We digested liver tissue in a 30% potassium hydroxide solution, and then glycogen was precipitated using 70% ethanol. After three washes (resuspend pellet in water, then add ethanol to 70% and centrifuge down pellet) to remove any traces of glucose, we digested glycogen by addition of amyloglucosidase. Released glucose was quantified using a colorimetric kit (Wako).
Stromal vascular fraction (SVF) and adipocyte isolation
We digested excised WAT in PBS containing 1% BSA and 1mg mL−1 type II collagenase for 30 minutes at 37°C with gentle agitation. The cell suspension was filtered through a 100 µm filter and then centrifuged at 700 × g for 5 minutes to separate floating adipocytes from SVF pellet. We washed floating adipocytes twice with PBS containing 1% BSA and collected the SVF pellets after each wash.
We homogenized tissues in lysis buffer (50mM Tris, pH7.5, 5mM EDTA, 250mM sucrose, 1% NP40, 2mM DTT, 1mM sodium vanadate, 100mM NaF, 10mM Na4
, and freshly added protease inhibitor tablet), and then incubated them for one hour at 4 °C 15
. We centrifuged crude lysates at 14,000 × g
for 15 minutes twice and determined the protein concentration using BioRad Protein Assay Reagent. Samples were diluted in sodium dodecyl sulfate (SDS) sample buffer. Bound proteins were resolved by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Bio-Rad). Individual proteins were detected with the specific antibodies and visualised on film using horseradish peroxidase-conjugated secondary antibodies (Bio-Rad) and Western Lightning Enhanced Chemiluminescence (Perkin Elmer Life Sciences). Alternatively, infrared fluorescent secondary antibodies were used for detection and quantification of specific protein on the Odyssey CLx imager (LI-COR).
Tissues were fixed in formalin for 3 days. The University of Michigan Cancer Center Research Histology Laboratory performed histology. A 1:100 dilution of the UCP1 (UCP11- Alpha Diagnostics) specific antibody at 10 µg mL−1 was used for immunohistochemical detection of UCP1 protein.
Gene expression analysis
We rinsed isolated mouse tissues in phosphate buffered saline (PBS), then froze them in liquid nitrogen and stored them at −80 °C until extraction. We extracted total RNA from Liver, WAT and BAT tissues as well as differentiated 3T3-L1 cells using the RNeasy Lipid Tissue Kit (Qiagen) according to the manufacturer’s instructions with the inclusion of a DNase digestion step. We extracted total RNA from BMDM and SVF cells using the RNeasy Kit (Qiagen) with a DNase step. We used the Superscript First-Strand Synthesis System for RT-PCR (Invitrogen) with random primers for reverse transcription. Realtime (RT) PCR amplification of the cDNA was performed on samples in triplicate with Power SYBR Green PCR Master Mix (Applied Biosystems) using the Applied Biosystems 7900HT Fast RT-PCR System. We chose Adrp and GAPDH as the internal control for normalization after screening several candidate genes; their expression was not significantly affected by experimental conditions. Sequences of all primers used in this study are listed in Supplementary Table 2
. Data was analyzed using the 2−ΔΔCT
method, and the control sample value was normalized to 1. Other data was quantified using an internal standard curve. Statistical significance was determined using the unpaired heterocedastic Student’s t-test with one averaged sample value per mouse.
Lipid oxidation rate
We excised intrascapular BAT and placed it in DMEM with 2% BSA with and without 50 µM amlexanox then incubated at 37 °C for 1 hour, after which we changed the media to DMEM with 2% BSA, 0.25 mM carnitine, 0.2 mM palmitic acid and trace levels of 3H-palmitic acid and incubated for one more hour at 37 °C and then collected the media, and isolated the aqueous faction. Lipid oxidation was determined by the conversion of 3H-palmitic acid to 3H2O.
IKKε and TBK1 in vitro kinase assays
We performed in vitro kinase assays by incubating purified kinase (IKKε or TBK1) in kinase buffer containing 25 mM Tris (pH7.5), 10 mM MgCl2, 1 mM DTT, and 10 µM ATP for 30 minutes at 30 °C in the presence of 0.5 µCi γ-[32P]-ATP and 1 µg MBP per sample as a substrate. We stopped the kinase reaction by adding 4x sodium dodecyl sulfate (SDS) sample buffer and boiling for 5 minutes at 95°C. Supernatants were resolved by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and analyzed by autoradiography using a Typhoon 9410 phosphorimager (GE Lifesciences). We quantified the bands using ImageQuant.
IKKε and TBK1 immune-complex kinase assay
We collected liver and white adipose tissues from C57BL/6 mice on ND or HFD, and homogenized the tissues using a Dounce homogenizer with lysis buffer containing 50 mM Tris (pH7.5), 150 mM NaCl, 2 mM EDTA, 5 mM NaF, 25 mM β-glycerophosphate, 1 mM sodium orthovanadate, 10% glycerol, 1% TritonX-100, 1 mM DTT, and 1 mM PMSF in the presence of protease inhibitors (Roche Diagnostics). We incubated tissue cell lysates for 1 hour at 4 °C and cleared them by spinning at 13,000 rpm for 15 minutes at 4 °C in a table-top centrifuge. Each 1 mg of lysate was subjected to immunoprecipitation using 5 µl of rabbit-polyclonal antibody against TBK1 or IKKε for 1.5 hours at 4 °C. We harvested immunocomplexes by incubation with ProtA beads (Roche Diagnostics) for 2 hours at 4 °C. We washed immunoprecipitates once with lysis buffer and three times with wash buffer containing 20 mM Hepes (pH 7.4), 50 mM NaCl, 20 mM β-glycerophosphate, 1 mM sodium orthovanadate, 5 mM NaF, 10 mM MgCl2, and 1 mM DTT. We performed an in vitro kinase assay using the immunoprecipitated kinases as described above. Relative levels of MBP phosphorylation were detected by autoradiograph and normalized to the levels of IKKε or TBK1 kinase detected in the immunoprecipitate by immunoblotting.
3T3-L1 fibroblasts (American Type Culture Collection) were cultured in 10% neonatal calf serum in Dulbecco’s modified Eagle medium (DMEM) for proliferation and 10% fetal bovine serum (FBS) in Dulbecco’s modified Eagle medium (DMEM) for differentiation. We grew the cells to confluence, then induced differentiation two days later with 500 µM 3-Isobutyl-1-methylxanthine, 250 nM dexamethasone, and insulin (1 µg mL−1) for three days, followed by insulin only treatment for two days. We routinely used cells within 7 days after completion of the differentiation process; and only used cultures in which >90% of cells displayed adipocyte morphology. We serum-starved 3T3-L1 adipocytes with 0.5% fetal bovine serum (FBS) in Dulbecco’s modified Eagle medium (DMEM) prior to treatment. TNF-α ng mL−1 unless otherwise noted) were performed during the 24 hour prior to harvest, after pretreatment with IKKβ inhibitor compound VIII (EMD Biosciences) for 1 hour where indicated. We pre-treated 3T3-L1 adipocytes for 1 hour with amlexanox at the given concentrations, then treated them with 20 µg ml−1 of poly I:C for 1 hour. Alternatively, we treated 3T3-L1 adipocytes with 50 µM forskolin for 15 minutes, after a 30-minute amlexanox pretreatment. We treated cells with or without 10 nM of insulin for 15 minutes. We determined glycerol release into the supernatant (DMEM without phenol red) using a colorimetric assay. We serum starved RAW264.7 cells with 0.5% FBS DMEM media and pre-treated with or without Cay-10576 (Cayman Chemical). The cells were then treated with LPS (0.5 µg ml−1) or poly I:C (50 µg ml−1) for 1 hour. We harvested the cells for total RNA and analyzed them by RT-PCR. We also resolved cell lysates by SDS-PAGE and analyzed them by immunoblot using the indicated antibodies. Wildtype and Ikbke/Tbk1 double knockout (DKO) and single knockout (SKO) MEFs were kindly provided by Dr. Shizuo Akira. Cells were cultured in DMEM with 10% FBS, and serum starved in 0.5% FBS for 12 hours prior to treatment. TNF-α treatment (50 ng mL−1) was performed for 15 minutes at the end of two hour pretreatment with amlexanox.
Oxygen consumption rate
The Michigan Metabolic Phenotyping Core and Michigan Nutritional Obesity Research Center measured oxygen consumption in cells and ex vivo tissue using an extracellular flux analyzer; model XF24-3 (Seahorse Bioscience).
Molecular modeling of amlexanox in the TBK1 ATP binding site
We modeled amlexanox into the known structure of the TBK1 catalytic domain (PDB ID codes 4EUT and 4EUU and 26
). The best fit of amlexanox in the active site of TBK1 was obtained using AutoDock Vina.
Averaged values are presented as the mean ± s.e.m. When comparing two groups, we determined statistical significance using the student’s t-test. When more than two groups were investigated, we first performed an ANOVA to establish that not all groups were equal. Following a statistically significant ANOVA, we performed between group comparisons using the Tukey-Krammer post-hoc analysis. ANOVA and Tukey-Krammer tests were performed using Stata version 12.0.