Supplemental methods
Rats
All rats were housed in individual cages with a 12/12 light dark schedule (lights on at
7.00 A.M.). Standard Rodent Chow and water were available ad libitum, unless stated
otherwise. All procedures were approved by the animal care committee of the Royal
Netherlands Academy of Arts and Sciences.
Tail incision
In the second week after arriving in the facility (week 0) a baseline blood sample was
taken. All blood samples in week 0 to 4 were taken by tail incision (1). On the days of
blood sampling, the rats were fasted from 09.00 A.M. and a blood sample was taken
~4 hours later between 1.00 and 2.00 P.M.
Denervation and jugular vein surgery
For both surgeries anaesthesia with 0.8 ml/kg Hypnorm i.m. (Janssen, High
Wycombe, Buckinghamshire, UK) and 0.4 ml/kg Dormicum s.c. (Roche, Almere, The
Netherlands) was used. One day after the baseline blood sampling in week 0, rats
received a sham, selective parasympathetic or sympathetic denervation (2). To
achieve a hepatic sympathectomy (Sx), nerve bundles running along the hepatic artery
proper were transected using microsurgical instruments. Any connective tissue
attachments between the hepatic artery, bile duct and portal vein were also dissected.
To achieve a hepatic parasympathectomy (Px), the common hepatic vagal branch was
transected by stretching the fascia containing the common hepatic vagal branch and
transecting neural tissue between the ventral vagal trunk and liver. Sham operated rats
underwent all procedures as described above, except for transection of the neural
tissue. The day after the tail vein blood sampling in week 4, the rats were fitted with
an intra-atrial silicone cannula into the right jugular vein according to the method of
Steffens (3). The cannula was tunneled to the head subcutaneously, fixed with dental
cement to four stainless steel screws inserted into the skull. A mixture of 60%
amoxicillin, 20% heparin, and 20% saline in polyvinylpyruvidon (PVP;Sigma) was
used to fill the cannulas and prevent inflammation and occlusion.
Measurement of VLDL-TG secretion
In week 5 rats were connected to an infusion swivel (Instech Laboratories, PA, USA)
on the day before the actual experiment, for adaption. One gram Tyloxapol (Triton
WR 1339; Sigma-Aldrich, Germany) was dissolved in 5.7 ml 0.9% saline overnight
on a shaker. On the next day rats were fasted from 09.00 A.M. onwards and external
lines for blood sampling were connected. At 1.00 P.M. a baseline blood sample from
the jugular vein was taken and an intravenous dosage of 0.7 ml Tyloxapol in saline
was given. At 20-min intervals blood samples were drawn from the jugular vein
catheter during 100 minutes. Tyloxapol inhibits lipoprotein lipase, thereby blocking
the uptake of triglycerides by the peripheral tissues. In the absence of chylomicrons
carrying triglycerides from the gut, the increase in plasma triglycerides reflects
VLDL-TG secretion (4). The VLDL-TG secretion (mmol/l/h) was determined by the
slope of the rise in triglycerides over time by linear regression analysis. A linear
increase in plasma triglycerides after Tyloxapol was shown by R2 values above 0.98
for all groups.
Tissue collection
In week 6 rats were fasted from 09.00 A.M. onwards and at 1.00 P.M. a blood sample
from the jugular vein was taken. Subsequently, rats were injected with an overdose of
pentobarbital IV. Liver tissue was removed and snap frozen for further analysis.
When the cannula was blocked, decapitation blood was collected after intraperitoneal
injection of pentobarbital.
Plasma measurements
Glucose concentrations were determined during the experiment in blood spots using a
glucose meter (FreestyleTM, Abbott, The Netherlands). Triglycerides and cholesterol
were assayed using a kit from Roche (Mannheim, Germany). The WAKO NEFA HR
kit (Wako. Chemicals, Neuss, Germany) was used to measure FFA in plasma. By
using radioimmunoassay kits, plasma insulin (LINCO Research, St. Charles, MO,
USA) and corticosterone (ICN Biomedicals, Costa Mesa, CA) were measured.
Liver measurements
The effectiveness of the hepatic sympathetic denervation was checked by
measurement of liver noradrenaline concentration using an in-house HPLC method
with fluorescence detection. Liver tissue samples of 50 mg were homogenized in 1 ml
of ice-cold NH4Cl buffer (0.2 M, containing 12 nmol/l -methylnorepinephrine and 1
g/l EDTA, pH 7.0) and centrifuged twice (14,000 rpm) for 15 min at 4°C. Essentially,
noradrenaline was selectively isolated by liquid-liquid extraction (5) and derivatized
with the fluorescent 1,2-diphenylethylenediamine (6). The fluorescent derivatives
were separated by reversed-phase liquid chromatography and detected by
fluorescence detection (510 pump, 717 plus autosampler, and 474 fluorescence
detector; Waters Chromatography). Separation of noradrenaline from other
endogenous compounds was achieved with a Waters Xterra RP18 column (5 m, 3.9
x150 mm). As an internal standard, -methylnorepinephrine was used. The detection
limit for noradrenaline was 0.05 nmol/l. All obese fa/fa Sx rats included in the
experiment showed liver noradrenaline concentrations below 1 ng/g (0,59±0,07;
mean±SEM) compared to obese fa/fa sham rats (18,41±4,03; mean±SEM). Three
obese fa/fa Sx rats were excluded based on higher liver noradrenaline concentrations.
Triglycerides in 50-100 mg liver tissue were measured after a single step lipid
extraction with methanol and chloroform (ratio 1:3) in a tissue-to-solvent ration of
1:80. (7). The pellets were finally dissolved in 2 % Triton X-100 (Sigma-Aldrich,
Germany) and triglycerides were measured using “Trig/GB” kit (Roche, Mannheim,
Germany). Liver acylcarnitines levels in 60 mg of liver tissue were measured
essentially as described previously (8).
Statistical analysis
Data are presented as mean ± SEM. For every experiment, two separate analyses were
performed. First, Fa/fa sham and fa/fa sham rats were compared by a repeated
measurements general linear model (GLM) for comparing outcome measures at
multiple time points (with Genotype as between-animal factor and Time as withinanimal factor) or a t-test for single outcome measures. Second, fa/fa sham, fa/fa Sx
and fa/fa Px rats were compared by repeated measurements GLM for comparing
outcome measures at multiple time points (with Denervation as between-animal
factor and Time as within-animal factor) or one-way ANOVA for single outcome
measures. A significant (P≤0.05) global effect of repeated measurements GLM or
ANOVA was followed by post hoc tests to detect individual group differences
(Fisher’s protected least significant difference). Significance was defined at P<0.05.
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