1.Animal model
Aldh2 *2/*2 knock-in mice were provided by the Dr. Che-Hong Chen from Dr. Daria
Mochly-Rosen’s lab, Stanford University. Aldh2 *2/*2 knock-in mice that on the
C57BL/C genetic background carry the Glu487Lys (E487K) were generated by
homologous recombination. All mice were housed in a temperature-controlled
environment at 23℃ under a 12 hr light/dark cycle. Mice were fed on
high-fat-high-sucrose diet (Cat. No. D12331, Research Diets, USA) or chow diet since the age of 4
weeks, respectively. Body weight was measured weekly. All animal experiments were
approved by the Institutional Animal Care and Use Committee of the Medical college of
National Taiwan University.
2.Glucose and insulin tolerance test
At 24 weeks, mice were evaluated by intraperitoneal glucose tolerance test (ipGTT)
after a 6-hour fasting at the age of 24 weeks. Tail blood glucose were measured at 0, 15,
30, 45, 60, 90, 120 min after intraperitoneal injection of glucose water (1mg/kg). The
insulin tolerance test (ITT) were conducted at the age of 25 weeks. Briefly, mice were
fasted 4 hours, and insulin (1.6IU/kg and 1.1IU/kg for HFHSD and chow diet respectively,
Humulin RTM, Eli Lily, USA) were injected intraperitoneally. Tail blood glucose were
measured at 0, 15, 30, 45, 60, 90, 120, 180 min. For oral glucose tolerance test (OGTT)
conducted at the age of 26 weeks, mice were fasted for 6 hours and glucose water (1mg/kg)
were given by oral gavage. Tail blood glucose was measured at 0, 15, 30, 60, 120 min
following glucose administration. Acute insulin response were measured using oral
glucose tolerance test (OGTT) at the age of 27 weeks. Mice blood was collected at 15 and
30 min during OGTT. Blood was centrifuged at 13000rpm, 10min, 4℃. Plasma was
collected for measurement insulin concentration.
3.Energy expenditure, food intake and physical activity
Indirect calorimetry measurements were performed using the Promethion Metabolic
Cage System (Promethion® , Sable Systems, Las Vegas, NV) at National Health Research
Institutes Laboratory Animal Center. VO2 and VCO2 of individual mouse were measured
for calculation of energy expenditure and the respiratory exchange ratio (RER). Wheels
in the chamber was used for measurement of the distance that mice run within 24 hours.
The food intake was measured for each mouse.
4. Cold tolerance test and diet-induced thermogenesis test
For a cold tolerance test, 10-week-old mice fasted for 4 hours were placed at 4℃
chamber and then the rectal temperature of mice was measured at 0, 1, 2, 3, 4 hour
respectively. Before the diet-induced thermogenesis test, 10-week-old mice fasted
overnight for 18 hours. The rectal temperature after HFHSD re-feeding were measured
at 0, 15, 30, 60, 120, 180, 240 min.
5.RNA extraction and RTqPCR
Brown adipose tissue from Aldh2 WT and KI mice was minced and harvested in 1ml
of REzol C&T (Cat. No. PT-KP200CT, Protech, Taiwan). 200μl of chloroform was added
to 1 ml of sample in Rezol, and samples were mixed vigorously by shaking for 30 seconds
and then were incubated at room temperature for 5 min. Then, samples were centrifuged
at 12000xg, 15min, 4℃, and 400 μl of the upper aqueous phase were transferred to a new
1.5 ml Eppendorf tube. An equal volume of isopropanol was added and samples were
inverted several times to mix and centrifuged at 12000xg, 10 min, 4℃. The RNA
precipitate, which formed a pellet at the bottom of 1.5 ml Eppendorf tube, was washed
with 75% ethanol for three times and are dried for 15 min. Pellets were dissolved the in
RNase-free water. The concentration of RNA was measured by Nanodrop. cDNA were
synthesized with reverse transcription kit (Cat. No. K1622, Thermo Scientific) by using
the oligo(dT)18 random primer. Real-time quantitative PCR (RT-qPCR) was performed in a 10 μl reaction with 50 ng cDNA and 0.4 μM primer using SYBR green reagent (Cat.
No. 1123ES08, YEASEN, China). Mouse Cyclophilin A mRNA was used as the internal
control. RT-qPCR reactions were performed by using ABI7900HT FAST (Applied
Biosystems, USA) and Sequence Detection Systems (SDS v2.3, Applied Biosystems).
All qPCR reactions were run in duplicates for each sample.
6.Primary cell culture
Brown adipose tissue from 4-6 weeks Aldh2 WT and KI mice were minced and
digested in 0.5% type I collagenase (Thermo Scientific) in 5 μM HEPES buffer for 40
min at 37℃. The digests were centrifuged at 600g for 5 min and the supernatant was
removed. The stromal vascular fraction (SVF) cell pellet was resuspended with PBS then
centrifuged at 600g for 5 min. After removal of the supernatant, pellet was resuspended
and cultured in 5μM Dulbecco’s modified Eagle medium: Nutrient Mixture F-12 (DMEM/F-12) (Cat. No. 12500062, Hyclone, USA) supplemented with 10% FBS (Cat.
No. 04-001-1A, Biological Industries, USA) and 1% antibiotic/antimycotic solution (Cat.
No. SV30079.01, Hyclone). For differentiation, primary brown preadipocyte were
exposed to induction medium containing 10% FBS, 0.5mM isobutyl-methylxanthine (Cat.
no. sc-201188A, Santa Cruz Biotechnology, USA), 1μg/ml insulin (Humulin RTM, Eli
Lilly), 5μM dexamethasone (Cat. no. D4902, Sigma-Aldrich, USA), 1nM T3(cat.no.
T5516, Sigma-Aldrich, USA), 125μM indomethacin (Cat. no. I7378, Sigma-Aldrich,
USA) as indicated. After 2 days, the medium was changed into DMEM/F12 containing
10% FBS, 1μg/ml insulin, and 1nM T3. Then we changed medium every 2 days until assay.
7.Western blot analysis
BAT homogenates from 30-week HFHSD Aldh2 WT and KI mice were prepared
using Radioimmunoprecipitation assay buffer (RIPA buffer) with protease inhibitor
cocktails (cOmplete™ ULTRA Tablets, Mini, EDTA-free, EASYpack Protease Inhibitor
Cocktail, Roche). The protein concentration was measured using the Bradford assay. We used 50 μg protein on the SDS-PAGE gel and then transferred it to the polyvinylidene
fluoride microporous membrane (GE Healthcare Life Science). The membranes were
blocked with 10% milk in PBS containing 0,1% Tween-20, and were probed with
antibodies UCP1 (Cat. no. GTX10983, GeneTex), 4HNE[HNEJ-2] (Cat. no. ab48506,
Abcam), Hsp70 (Cat. no. ab45133, Abcam), GAPDH (Cat. no. MAB374, Merck).
Corresponding secondary antibodies conjugated to horseradish peroxidase were used the
chemiluminescence. Signal were detected using TOPBIO Chemiluminescence image
system (MultiGel-21, MGIS-21-C2-6M).
The protein using for detecting with biotin hydrazide and 4HNE antibody was
incubated in 10mM NaBH4 for 30min. For carbonylated proteins, samples chemically
reduced by NaBH4 were then incubated with 5mM EZ-link biotin hydrazide (Cat. no.
21339, Pierce) for 1 hour. After coupling, the samples are separated by SDS-PAGE gel
and transferred to the polyvinylidene fluoride microporous membrane (GE Healthcare
Life Science). The membrane was blocked with 10% milk in PBS containing 0,1%
Tween-20 overnight at 4℃. Rinsed with PBST and incubated with Streptavidin-HRPA (Cat. no. 890803, BD Biosciences) for 1hour. Signal were detected using TOPBIO Chemiluminescence image system (MultiGel-21, MGIS-21-C2-6M).
8.Fatty acid oxidation assay
Differentiated primary brown adipocytes from Aldh2 WT and KI mice cultured in
24-well-plate were washed with PBS three times, and incubated with 250μl
palmitate/BSA buffer including 0.5 μCi [3H]-palmitate (Cat. no. PK-NET043001MC,
3H-Palmitic acid,1mCi, PerkinElmer) for 2 hours. At the end of the incubation period,
the reaction mixture was transferred to the 2ml Eppendorf tube. We then add 750μl chloroform, 250μl chloroform/methanol (1:2 volume mixture), and 250μl 2M KCl/HCl to previous 2ml Eppendorf tube in sequence. The samples were vortexed for 5 seconds
and centrifuged at 3500rpm, 10min, 4℃. 600μl upper aqueous phase was transferred to
new 2 ml Eppendorf tubes. 400 μl chloroform was then added and vortexed for 5 seconds, followed by addition of 400 μl methanol. Then 360 μl 2M KCl/HCl was added and vortexed for 5 seconds. The samples were centrifuged at 3500rpm, 10min, 4℃. 1ml upper
aqueous phase was transferred to scintillation vial which contains 4 ml of scintillation
fluid and was measured the average counts per minute (CPM) by a liquid scintillation
counter.
Regarding fatty acid oxidation of tissues, Aldh2 KI and WT mice were fed with
HFHSD for 5 weeks. Mouse BAT was isolated, weighed, and added STE buffer (0.25M
Sucrose,10mM Tris-HCl, 1mM EDTA, pH7.4), which the volume we added was
100mg/ml of tissue. BAT was homogenized using a glass Dounce homogenizer with the
“Loose” pestle down and up 10 strokes each. The homogenate was poured to 1.5 ml
Eppendorf tube and centrifuged at 500g for 10min at 4℃. The supernatant was decanted
to new 1.5ml Eppendorf tube. We transferred 30 μl of tissue homogenate to 24-well-plate containing 370μl reaction mixture buffer which had prepared previously. Each sample
was running in triplicate. The reaction mixture buffer was prepared with 100mM sucrose,
10mM Tris-HCl, 5mM KH2PO4, 0.2mM EDTA, 80mM KCl, 1mM MgCl2, 2mM
L-carnitine, 0.1mM malate, 0.05mM Coenzyme A, 2mM ATP, 1mM DTT, 7% BSA/5mM palmitate/ 0.01 μCi/μl [3H]-palmitate. The samples were incubated for 60min at 37℃. At
the end of the incubation, the total volume of each sample was transferred to 2ml Eppendorf tube. We then add 750μl chloroform, 250μl chloroform/methanol (1:2 volume
mixture), and 250μl 2M KCl/HCl in sequence. The samples were vortexed for 5 seconds and centrifuged at 3500rpm, 10min, 4℃. 600μl upper aqueous phase was transferred to
new 2 ml Eppendorf tubes. 400 μl chloroform was then added and vortexed for 5 seconds, followed by addition of 400 μl methanol. Then 360 μl 2M KCl/HCl was added and vortexed for 5 seconds. The samples were centrifuged at 3500rpm, 10min, 4℃. 1ml upper
aqueous phase was transferred to scintillation vial which contains 4 ml of scintillation
fluid and was measured the average counts per minute (CPM) by a liquid scintillation
counter.
9.Isolation of brown adipose tissue mitochondria
The BAT from 30-week-old Aldh2 KI and WT mice were homogenized in the
Dounce homogenizer with ice-cold mitochondrial isolation buffer (210mM Mannitol,
70mM sucrose, 1mM EGTA, 5mM HEPES pH7.5, 0.5%BSA), which the volume we
added was 100mg/ml of tissue. The homogenate was centrifuged 800g for 10 min at 4℃
and the supernatant was decanted and filtered through two-layer cheesecloth to remove
residual particulates. The filtered homogenate was centrifuged by 8000g for 10 min at
4℃ and carefully removed the supernatant. The pellet was resolved with 1ml
mitochondrial isolation buffer and centrifuged again. The mitochondrial pellet was re-suspended in 30 μl of STE buffer (0.25M Sucrose,10mM Tris-HCl, 1mM EDTA, pH7.4)
and protein concentration was quantitated using the Bradford assay.
10.In-solution digestion
The proteins samples in 6 M urea were reduced by 20 mM dithiothreitol for 1 h at
60°C, and alkylated by 55mM iodoacetamide for 45 min in the dark at room temperate.
Then, the samples were diluted 6-fold with 50 mM triethyl ammonium bicarbonate buffer
and digested with trypsin or chymotrypsin at an enzyme:protein ratio of 1:50 w/w for
16-18 h at 37 °C and 25 °C individually. Digestion was quenched by an addition of
trifluoroacetic acid to a final concentration of 1%. Peptides were desalted with C18 ziptip
(Millipore Corp., Billerica, MA, USA) according to the instructions of the manufacturer.
Desalted and dried peptides were resuspended in 10μL of 0.1% formic acid (FA), and
ready for LC-MS/MS analysis.
11.LC-MS/MS analysis
LC-MS/MS analysis was performed on a NanoACQUITY UPLC System (Waters,
USA) coupled to a high-resolution mass spectrometer (QE HF-X, Thermo Fisher Scientific, USA). The peptides were injected into a trap column (2 cm × 75 μm i.d.,
Symmetry C18), then separated in a 25 cm × 75 μm i.d. BEH130 C18 column (Waters,
USA) by a gradient from 0% to 85% buffer B (buffer A, 0.1% formic acid in H2O; buffer
B, 0.1% formic acid in acetonitrile). The mass spectrometer is operated in data-dependent
mode with the following acquisition cycle: a MS scan (m/z 350–1600) recorded at
resolution R = 60,000; MS/MS scans recorded at resolution R = 15,000, which are
acquired by HCD fragmentation with collision energy of 28.
12.Database analysis
MS/MS spectra were searched with the Mascot engine (v2.6, Matrix Science, UK)
against the UniProtKB mouse protein database using the following parameters: the mass
tolerance of precursor peptide was set as 20 ppm, and the tolerance for MS/MS fragments
was 0.02 Da with maximum two missed cleavage. The modifications of peptides were set
as follows: static carbamidomethylation on cysteine, variable oxidation on methionine,
variable deamidation of asparagine or glutamine, and variable 4-HNE modification on
cysteine, histidine and lysine. The cut-off threshold of significant peptide-to-spectrum
matches is p < 0.05.
Mitochondrial modified proteins biological function were confirmed by using the Uniport database. Then we used g:Profiler to get the “Kyoto Encyclopedia of Genes and
Genomes (KEGG) pathways” and “Gene ontology biological process” information.