SUPPLEMENTAL METHODS
Caloric Intake. Caloric intake was calculated daily as grams of food eaten multiplied by the
caloric content of each diet (Western diet: 4.5 kcal/g of diet and Chow: 3.1 kcal/g diet). These
data were then normalized to body weight. The total fat intake per day, based on 90% absorption
(Figure 1C) was 218 mg/d in chow-fed mice and 954 mg/d in either western- or western plus
naringenin-fed mice.
Blood and tissue collection. Blood for metabolic studies was taken via the saphenous vein. Mice
were sacrificed by CO2 inhalation, blood was obtained by cardiac puncture and plasma stored at 20°C. Animals were dissected down the midline and tissues excised, weighed, snap frozen and
stored at -80°C.
Tissue histology. Liver was dissected, mounted in OCT, frozen and sectioned using a Leica
CM3050S cryostat. Tissue sections were fixed in 4% paraformaldehyde and stained with Oil Red
O and haematoxylin. Pancreas, white adipose tissue (WAT) and brown adipose tissue (BAT)
were fixed in 10% formalin, paraffin embedded and sections made on a Microm HM335E
Microtome. WAT was stained for Mac-1 as previously described (1). Photomicrographs were
obtained using an Olympus BX50 microscope and a QImaging Retiga EXi FAST camera.
TG and cholesterol absorption. Each mouse was gavaged with 150 μL of medium chain
triglyceride (MCT) oil (Novartis Medical Nutrition, Fremont, MI) containing 2.5 μCi of [C 14]Triolein (Amersham, Piscataway, NJ) or 1 μCi of [4-C14]-Cholesterol (Perkin Elmer) and 2.5 μCi
of [5,6-H3]-β-sitostanol (American Radiolabeled Chemicals Inc., St Louis. MO). Mice were then
placed individually in metabolic cages for 48 h, feces collected every 24 h and extracted. The
ratio of [C14] to [H3] was used to calculate the percentage of lipid absorbed (2, 3). In all animals,
the recovery of radiolabeled sitostanol was 75-90%. The data are represented as the mean
percent lipid absorption in each group over 48 hours.
TG and apoB100 secretion. Mice were fasted for 6 h before intra-peritoneal (i.p.) injection with
1 g/kg Tyloxapol U.S.P. (0.15 g /ml in 0.9% NaCl) (Ruger Chemical Company, Irvington, NJ).
Following i.p. injection, tyloxapol takes between 30-60 minutes to be absorbed into plasma.
Tyloxapol coats lipoproteins preventing TG hydrolysis and VLDL clearance. Under these
conditions, tyloxapol blocks the catabolism of TG-rich lipoproteins derived from both the liver
and intestine (4, 5). Samples were collected at 0, 30, 60 and 120 min post-injection by cardiac
puncture for plasma TG measurement. For apoB secretion, mice were injected i.p. with 200 μCi
of Tran 35S-label (1000 Ci/mmol, L-[35S]-methionine and L-[35S]-cysteine, MP Biomedicals Inc.,
Irvine, CA) in addition to Tyloxopol and sacrificed at 60 and 120 min (6). A combined VLDL
and IDL fraction was isolated from 200 μl of plasma by ultracentrifugation (Beckman TLA120.2; 100,000 rpm, 6 h at d < 1.019 g/ml). The VLDL/IDL fractions were subjected to 4.5%
SDS-PAGE, the gel dried and exposed to a phosphorimager screen. The volumes of apoB48 and
apoB100 bands were determined using ImageQuant software (Molecular Dynamics, Sunnyvale,
CA) (7).
Heparin releasable LPL activity (LPLA). Mice were injected i.p. with 100 U/kg heparin
(Pharmaceutical Partners of Canada, Richmond Hill, ON) and sacrificed 30 min later. Fifty μL of
plasma was assayed for total lipase activity assay (8) and hepatic lipase activity which was
determined using the same assay with the addition of 1 M NaCl and 0.4 mM Tris at pH 8.9 to all
buffers. LPL activity was calculated as the difference between total and hepatic lipase activities
(9).
Fatty acid oxidation. Briefly, 250 mg of fresh tissue was homogenized in 0.1 M phosphate
buffer containing 0.25 M sucrose and 1 mM EDTA using a Potter-Elvehjem homogenizer.
1
Tissue homogenates were incubated for 30 minutes at 37ºC in a buffer containing 150 mM KCl,
10 mM Hepes pH 7.2, 5 mM Tris malonate, 10 mM MgCl2, 1 mM carnitine, 0.15% fatty acid
free bovine serum albumin (FAF-BSA), 5 mM ATP and 50 μM 3H-palmitate complexed with
FAF-BSA. Reactions were stopped with addition of 200 uL 0.6 N perchloric acid. Unreacted
fatty acids were extracted with n-hexane and 3H2O measured by liquid scintillation counting
(10).
Fatty acid and cholesterol synthesis. Fatty acid and cholesterol synthesis were measured
following i.p. injection of [1-14C]-acetic acid (Amersham, GE Healthcare, UK). Briefly, mice
were injected with 20 µCi of acetic acid and sacrificed 30 minutes later. Tissues (500 mg) were
extracted in chloroform:methanol and incorporation of 1-14C acetic acid into fatty acid and
cholesterol were assayed as described previously (8).
Gene expression by qRT-PCR. Tissue mRNA levels were determined by quantitative real-time
RT-PCR as published previously (11). Expression of Aco, Acat1/2 (Soat1/2), Cpt1α, Cpt1β,
Dgat1, Mttp, Pgc1α, Pparα, Srebf1c (Srebp1c), Ucp1, and Ucp3, were normalized to Gapdh
expression. The primer and probe sets were obtained from Taqman® Assays-on-Demand
(Applied Biosystems) except Srebf1c which was custom designed from the sequence of Mus
musculus Srebf1 (Genbank accession no. AL669954). Primers and probe for Srebp1c were
designed such that the probe overlapped the boundary between exon-1 specific for Srebp1c and
exon 2 (Applied Biosystems). Forward Primer: CAGGCCCGGGAAGTCACT, Reverse Primer:
GACCACGGAGCCATGGATT, Probe: ATTTGAAGACATGCTCCA (Gene is on the minus
strand and primers have been designed as such).
mtDNA measurement. Liver (10mg) was homogenized and DNA was extracted by the
Puregene DNA purification system (Qiagen) as per manufacturer’s instructions and quantitative
real-time PCR was performed using TaqMan technology (ABI Prism7700 Sequence Detection
System). Primer and probe sequences, specific to the mitochondrial D-loop region and nuclear
genomic telomerase region were as previously described (12).
Glucose and insulin tolerance tests (GTT and ITT). Mice were injected i.p. with 15% glucose
in 0.9% NaCl (1 g/kg of body weight). Blood for glucose measurements (Ascensia Elite
glucometer, Bayer Healthcare, Toronto, Canada) was taken up to 180 min post-injection. Insulin
tolerance tests (ITT) were conducted by i.p. injection with 0.5 IU/kg Humulin (Eli Lily). Blood
for glucose measurements was obtained up to 60 min post-injection. Insulin sensitivity and
glucose utilization and were calculated based on the area under the curve (AUC) and the
incremental AUC (IAUC).
A GTT in C57BL6/J mice was performed following gavage of 20% glucose in 0.9% NaCl (1
g/kg body weight) and blood glucose measurements taken as described above. An ITT was
performed following an i.p. injection with 1 IU/kg Humulin (Eli Lily). Blood for glucose
measurements was taken as described above.
Glucose uptake assay. Mice were injected i.p. with 12 μCi/mouse of 2-deoxy-D-[1-3H]-glucose
(Amersham, GE Healthcare, UK) and with 1 g/kg cold glucose under the conditions described
for the GTT and deoxyglucose uptake determined after 1h as described previously (13) with the
addition of 14C-deoxyglucose as a recovery standard.
Energy Expenditure. Briefly, mice were placed in cages for 24 h with free access to food and
water. Every 15 min, O2 and CO2 concentrations were measured in each chamber and O2
consumption and CO2 production calculated. Data for EE were adjusted for body weight (14).
2
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