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Neural control of hepatic lipid metabolism: A (patho)physiological perspective
Bruinstroop, E.
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Citation for published version (APA):Bruinstroop, E. (2013). Neural control of hepatic lipid metabolism: A (patho)physiological perspective
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Download date: 20 Jun 2018
SYMPathEtic and ParaSYMPathEtic hEPatic
dEnErVatiOn MOdulatE dYSliPidEMia in ZucKEr ratS 5
Eveline Bruinstroop, jitske Eliveld, Ewout foppen, Sander Busker, Mariette t. ackermans, Sander M. houten,
Eric fliers and andries Kalsbeek
ABSTRACT
Human and animal studies increasingly point towards a neural pathogenesis of the
metabolic syndrome, involving hypothalamic and autonomic nervous system dysfunction.
We hypothesized that increased VLDL-Triglyceride (VLDL-TG) secretion by the liver in the
obese Zucker (fa/fa) rat, is due to a relative hyperactivity of sympathetic or hypoactivity
of parasympathetic hepatic innervation. To investigate this, we surgically denervated the
sympathetic or parasympathetic hepatic nerve in obese Zucker rats. Our results show
that cutting the sympathetic hepatic nerve lowers VLDL-TG secretion, finally resulting in
lower plasma triglyceride concentrations. By contrast, a parasympathetic denervation
results in increased plasma total cholesterol concentrations. The effect of a sympathetic or
parasympathetic denervation of the liver was independent of changes in humoral factors or
changes in body weight or food intake. In conclusion, a sympathetic denervation improves
the lipid profile in obese Zucker rats with marked dyslipidemia, whereas a parasympathetic
denervation increases total cholesterol levels.
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INTRODUCTION
Increasing evidence in humans suggests an important role for the autonomic nervous
system in the development of obesity, diabetes and dyslipidemia (1-3). These studies
showed that increased sympathetic and decreased parasympathetic drive correlate with
several factors of the metabolic syndrome, including hypertriglyceridemia. In the field of
hypertension, the autonomic nervous system was regarded a treatment target already in the
1930s, when the first renal denervation was carried out (4). Due to major pharmacological
discoveries the surgical strategy has been not used for many decades but is now again
being investigated as a treatment of resistant hypertension in humans (5).
We therefore aimed to investigate in the present study if increased secretion of VLDL-TG,
which is a major contributor to dyslipidemia, can be reduced with a surgical denervation of
the liver in an animal model of dyslipidemia. The obese Zucker rat (fa/fa) has high triglyceride
and cholesterol plasma levels, which are already apparent shortly after weaning, due to an
overproduction of VLDL-TG by the liver (6-9). In contrast to reports of low sympathetic nerve
activity to brown adipose tissue, obese Zucker rats show high levels of renal sympathetic
nerve activity and elevated baseline greater splanchic nerve activity, suggesting increased
sympathetic nervous activity to the liver (10,11).
Previous studies in rats showed mounting evidence for the involvement of the autonomic
nervous system in the secretion of triglycerides packed in VLDL particles (VLDL-TG) from
the liver (12,13). These studies led us to believe that a sympathetic denervation of the liver
may be beneficiary in reducing VLDL-TG secretion.
Based on the human studies showing autonomic dysfunction in dyslipidemia and rat
studies showing effects of the autonomic nervous system on VLDL-TG secretion by the
liver we hypothesized that a sympathetic denervation should have a beneficiary effect on
plasma triglycerides and VLDL-TG secretion by the liver in obese Zucker rats. Conversely, a
parasympathetic denervation might have an opposite effect. We tested our hypothesis in
lean (Fa/fa) and obese (fa/fa) Zucker rats by following plasma TG levels and hepatic VLDL-TG
secretion during a timeframe of six weeks after a surgical sympathetic or parasympathetic
denervation of the liver.
MATERIAL AND METHODS
Zucker rats
In total 31 obese Zucker rats (fa/fa; Charles River Germany) were included in the experiment
and received a hepatic sham denervation (fa/fa sham n = 10), hepatic sympathetic denervation
(fa/fa Sx n = 12) or a hepatic parasympathetic denervation (fa/fa Px n = 9). For comparison,
7 age matched lean Zucker rats with a heterozygous mutation (Fa/fa) were included and
received a sham denervation (Fa/fa sham n = 7). 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. Body weight, food intake and water
intake were measured on all weekdays. All procedures were approved by the animal care
committee of the Royal Netherlands Academy of Arts and Sciences.
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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 (14). 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. Hepatic sympathetic or parasympathetic
branches were denervated according to previous reports (15). The day after the blood
sampling in week 4, the rats were fitted with an intra-atrial silicone cannula into the right
jugular vein (16,17).
Measurement of VlDl-TG secretion
In week 5 rats were connected to an infusion swivel (Instech Laboratories, PA, USA) one
day before the experiment for adaption. 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 15%
tyloxapol (Sigma-Aldrich, Germany) was given. At 20-min intervals blood samples were
drawn from the jugular vein catheter during 100 minutes.
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, adipose tissue and the brain were 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 VLDL-TG secretion rate was
determined by the slope of the rise in triglycerides over time by linear regression analysis.
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
Triglycerides in liver were measured after a single step lipid extraction with methanol
and chloroform (18). The pellets were finally dissolved in 2 % Triton X-100 (Sigma-Aldrich,
Germany) and triglycerides and cholesterol were measured (Roche, Mannheim, Germany).
The effectiveness of the hepatic sympathetic denervation was checked by measurement of
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liver noradrenaline concentration using an in-house HPLC method with fluorescent detection
(17). RT-PCR was performed as previously described with the same primer sequences (13).
Liver acylcarnitines levels were measured essentially as described (19).
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 within-animal 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.
RESULTS AND DISCUSSION
Selective hepatic denervation of parasympathetic and sympathetic nerve
To investigate the effect of a selective sympathetic or parasympathetic hepatic denervation
on the development of dyslipidemia in the obese Zucker rat (fa/fa), we surgically cut
either the sympathetic (Sx) or parasympathetic (Px) branch to the liver and compared
these groups with sham denervated rats. To evaluate the effect size, we included sham
denervated heterozygous lean Zucker rats (Fa/fa) in the experiment. For the fa/fa Sx rats, a
successful denervation was defined as achieving a liver noradrenaline concentration after
the experiment of below 1 ng/g of liver tissue. All fa/fa Sx rats, except three, showed liver
noradrenaline concentration below 1 ng/g (0,59 ± 0,07; mean ± SEM) compared to fa/fa
sham rats (18,41 ± 4,03; mean ± SEM). We have previously validated our method for selective
hepatic parasympathectomy by using retrograde viral tracing (15). During a timeframe of
seven weeks, all rats were included in a protocol with weekly blood sampling by tail incision
(week 0-4), a VLDL-TG secretion experiment in week 5 and a final blood sample and tissue
collection (week 6) (Figure 1A). Additionally, daily measurements of body weight, food intake
and water intake were undertaken.
A sympathetic denervation lowers VlDl-TG secretion and plasma triglycerides
Obese fa/fa sham rats show significantly higher plasma triglyceride concentrations compared
to lean Fa/fa sham rats at all time points (week 0-4: Time p = 0.032, Time*Genotype
p = 0.123, Genotype p = 0.001; week 5 and 6 p < 0.01) (Figure 1B). When comparing the
Sham, Sx and Px fa/fa groups, a significant 39% decrease in plasma triglycerides was found
in the fa/fa Sx rats compared to fa/fa sham (p = 0.040) and 56 % compared to fa/fa Px
(p = 0.001) rats in week 6. It is well known that obese Zucker rats show increased lipoprotein
lipase (LPL) activity in white adipose tissue, which partly compensates for the increase in
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Fig
ure
1.
A s
ymp
ath
eti
c d
en
erv
atio
n lo
we
rs v
LDL-
TG s
ecr
eti
on
and
pla
sma
trig
lyce
rid
es
and
a p
aras
ymp
ath
eti
c d
en
erv
atio
n in
cre
ase
s p
lasm
a to
tal
cho
lest
ero
l in
ob
ese
Zu
cke
r ra
ts. (
A) G
rap
hica
l rep
rese
ntat
ion
of s
tud
y d
esig
n (B
) Pla
sma
trig
lyce
rid
e m
easu
rem
ents
dur
ing
the
exp
erim
ent (
* p
< 0
.05
com
par
ed
to fa
/fa
Sham
) (C
) VLD
L-TG
sec
reti
on
rate
s ca
lcul
ated
fro
m t
he in
crea
se o
f pla
sma
trig
lyce
rid
es a
fter
inje
ctio
n o
f tyl
oxa
po
l (D
) Pla
sma
tota
l cho
lest
ero
l mea
sure
d
in t
he fi
nal b
loo
d s
amp
le (E
) Bo
dy
wei
ght
of a
ll g
roup
s d
urin
g w
eek
0-6
(F) A
vera
ge
dai
ly fo
od
inta
ke o
f all
gro
ups
dur
ing
wee
k 0
-6. V
alue
s ar
e m
ean
± S
EM o
f 6
-10
rats
per
gro
up. *
P <
0.0
5.
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VLDL-TG secretion (20,21). In week 5 VLDL-TG secretion by the liver was therefore measured
after injection of tyloxapol. Tyloxapol inhibits LPL-mediated triglyceride hydrolysis and thus
prevents the removal of circulating triglycerides (8,22). We found that VLDL-TG secretion
is more than twice as high in fa/fa sham rats compared to Fa/fa sham rats (p < 0.001;
Figure 1C). Furthermore, this experiment showed lower VLDL-TG secretion rates in the fa/fa
Sx rats compared to fa/fa sham (p = 0.026) and fa/fa Px (p = 0.012) rats before any changes in
plasma TG concentrations occurred. Apparently, the clearance of triglycerides in peripheral
tissues masked the effect of the reduced VLDL-TG secretion on plasma triglycerides in
week 0-5. In fa/fa Sx rats, plasma total cholesterol concentrations showed no difference
compared to fa/fa sham rats.
A parasympathetic denervation increases plasma total cholesterol concentrations
Obese fa/fa sham rats showed significantly higher plasma total cholesterol concentrations
compared to lean Fa/fa sham rats at all time points (week 0-4: Time p = 0.081, Time*Genotype
p = 0.487, Genotype p < 0.001; week 5 and 6 p < 0.01). In week 6 a significant increase
of total cholesterol in fa/fa Px rats compared to fa/fa sham (p = 0.045) and fa/fa Sx rats
(p = 0.002) was observed (Figure 1D). It is interesting to observe that the effects of a
parasympathetic denervation are very different from a sympathetic denervation, with an
increase of plasma triglycerides in week 5 (Figure 1B) and increased total cholesterol levels in
the final sample. Human studies have also indicated that contrary to high sympathetic activity,
low parasympathetic activity is correlated to dyslipidemia (2). To our knowledge, we are the
first to investigate the effects of a sympathetic denervation to the liver in Zucker rats, although
surgical parasympathetic denervations in Zucker rats have been performed previously (23,24).
Ferrari et al., (24) investigated the effect of a subdiaphragmatic vagal deafferentation (SDA),
cutting all vagal afferents from the gut, including the liver, and leaving half of the vagal
efferents intact. Contrary to our results from a selective hepatic vagal denervation they found
decreased body weight gain and food intake, decreased plasma insulin and triglyceride
concentrations and reduced total liver fat content in obese Zucker rats treated with SDA. This
indicates that the effects of the SDA are caused by a liver independent mechanism, as we did
not observe any of these effects after a selective parasympathetic denervation of the liver.
Hepatic denervations do not influence body weight, food intake and water intake
As a sympathetic denervation lowered plasma triglycerides and a parasympathetic
denervation elevated total cholesterol levels, we investigated whether these changes could
be attributed to changes in body weight gain, food intake or water intake. All groups
significantly increased their body weight during the experiment (Figure 1E). Obese fa/fa
sham rats were heavier at baseline and showed increased body weight gain during the
experiment compared to lean Fa/fa sham rats (Time p < 0.001, Time*Genotype p < 0.001,
Genotype p < 0.001). On average obese fa/fa rats gained 117 ± 7 grams of body weight
during the experiment, whereas lean Fa/fa rats gained 61 ± 3 grams. Comparison between
fa/fa sham, fa/fa Sx and fa/fa Px rats revealed no significant differences in body weight
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during the experiment (Time p < 0.001, Time*Denervation p = 0.334, Denervation p = 0.857).
Average food intake during the study was significantly increased in fa/fa sham rats compared
to Fa/fa sham rats (p < 0.001), but no differences were observed between the fa/fa groups
(Figure 1F). Water intake was not significantly different for any of the groups. Therefore we
conclude that the above described changes occurred independent of changes in body
weight gain, food intake and water intake.
Hepatic denervations do not influence insulin, glucose, FFA and corticosterone plasma levels
VLDL-TG secretion is regulated by the availability of nutrients (e.g., FFA and glucose)
and hormones (e.g., insulin and corticosterone). Obese Zucker rats are hyperinsulinemic
in the face of unchanged plasma glucose concentrations and chronic hyperinsulinemia
is known to increase VLDL-TG secretion. Studies have shown differences in plasma FFA
and corticosterone levels in obese versus lean Zucker rats. To investigate if the changes
in VLDL-TG secretion after a sympathetic denervation can be attributed to these factors
blood glucose, plasma insulin, corticosterone and free fatty acids (FFA) were measured
in the final sample. As expected, plasma insulin was significantly higher in fa/fa sham rats
(7,71 ± 0,54; mean ± SEM) compared to Fa/fa sham rats (1,12 ± 0,13; mean ± SEM). Of note,
no significant differences were observed between the fa/fa sham, fa/fa Sx and fa/fa Px rats.
Furthermore, blood glucose, plasma corticosterone and plasma FFA were not significantly
different between fa/fa sham and Fa/fa sham rats, nor between the fa/fa denervation
groups. Therefore, these humoral factors do not explain the changes observed in fa/fa Sx
and Px rats and support a direct control of autonomic nerves on VLDL-TG secretion.
fa/fa Sx rats do not display changes in the expression of key enzymes involved in lipogenesis, VlDl-TG secretion and beta-oxidation
To further dissect the hepatic mechanism responsible for the changes observed in plasma
triglycerides, we measured liver triglyceride concentration and the mRNA expression profile
of key enzymes involved in lipid metabolism. Obese fa/fa sham rats showed over 4-fold
higher triglyceride levels in the liver compared to lean Fa/fa sham rats (p = 0.002). However,
no differences were observed between the fa/fa sham, fa/fa Sx and fa/fa Px rats (Figure 2A).
Therefore, we can conclude that the lower rate of triglyceride secretion by the sympathetically
denervated liver does not result in an increased accumulation of triglycerides in the liver. Liu
et al. (25) investigated the effect of a pharmacological blockade of the sympathetic nervous
system for seven days with carvedilol in an animal model of alcohol fatty liver disease and
found that this attenuates the progression of steatosis. Our data show that this is not the
case with surgical sympathetic blockade in a model of non-alcoholic fatty liver disease. To
further investigate if lipogenesis and TAG storage is changed in fa/fa Sx rats we measured
mRNA levels of key genes in this pathway. It is known that the rate of lipogenesis is higher
in obese fa/fa rats, secondary to the hyperphagia and hyperinsulinemia (26,27). Increased
lipogenesis was also shown in our experiments by the increased mRNA expression of Fas
(Figure 2B). However, no differences were observed between the denervation groups,
showing that the lower VLDL-TG secretion rates in sympathetically denervated rats are not
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Fig
ure
2. O
be
se Z
uck
er
rats
sh
ow
hig
he
r liv
er
trig
lyce
rid
es
con
cen
trat
ion
s, h
igh
er
mR
NA
exp
ress
ion
of Fas
and
low
er
leve
ls o
f lo
ng
-ch
ain
acyl
carn
itin
es.
(A
) Liv
er t
rig
lyce
rid
e co
ncen
trat
ions
(B
) Liv
er m
RN
A e
xpre
ssio
n o
f ke
y en
zym
es in
hep
atic
lip
id m
etab
olis
m (C
) Liv
er a
cylc
arni
tine
s co
ncen
trat
ions
. Va
lues
are
m
ean
± S
EM o
f 6-1
0 ra
ts p
er g
roup
. * P
< 0
.05,
**
P <
0.0
1.
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due to a decreased expression of FAS resulting in lower lipogenesis rates. mRNA levels of
other genes involved in lipogenesis (Acc1, Acc2, Scd1, Srebp1c, Pparγ) were not affected
by the genotype or the denervation. Apart from effects on lipogenesis, we measured
mRNA expression of genes involved in the assembly and secretion of VLDL-TG, but no
difference in the mRNA levels of Arf1, ApoB, Mttp and Dgat1 was observed between the
fa/fa sham and Fa/fa sham rats, nor was any effect of the denervation in the fa/fa groups
observed. Finally, we investigated if fa/fa Sx rats increase their levels of fatty acid beta-
oxidation, leaving less substrate for VLDL-TG secretion. It is known that obese Zucker rats
favor triglyceride synthesis over fatty acid oxidation and are less capable to oxidize fatty
acids in the liver (9). To this end, we measured mRNA levels of Cpt1a but found no significant
differences between the Fa/fa and fa/fa groups or the different denervation groups. We also
measured the levels of acylcarnitines in the liver to rule out any effect on beta-oxidation.
We observed that most long-chain acylcarnitines were decreased, which is consistent with
lower beta-oxidation in obese Zucker rats when compared to lean Zucker rats (Figure 2C).
However, fa/fa Sx rats do not display elevated levels of these acylcarnitines and therefore
beta-oxidation seems unaffected in the denervated rats. From these experiments we can
conclude that decreased lipogenesis or TG accumulation and increased beta-oxidation
within the liver cannot explain lower VLDL-TG secretion in fa/fa Sx rats. Alternatively, the
sympathetic nervous system may regulate lipid metabolism by other mechanisms, e.g. by
altering hepatic blood flow. Further studies are necessary to reveal these mechanisms.
CONCLUSIONS
In this study we confirm the hypothesis that cutting the sympathetic nerve to the liver lowers
VLDL-TG secretion and results in subsequent lower plasma triglycerides in obese Zucker
rats. On the contrary, a parasympathetic denervation results in increased plasma cholesterol
and triglyceride concentrations. The effects of the sympathetic and parasympathetic
hepatic denervations were independent of changes in body weight, food intake, water
intake, plasma glucose, FFA, insulin and corticosterone. Thus in addition to hyperphagia
and hyperinsulinemia, the autonomic nervous system is an independent determinant of
VLDL-TG secretion and plasma cholesterol in obese Zucker rats. Therefore, in addition to
caloric restriction and treatment of hyperinsulinemia in order to reduce hypertriglyceridemia
selective hepatic denervations may be a useful intervention to modulate lipid metabolism.
ACkNOwLEDGEMENTS
This work is supported by NWO-ZonMw (TOP 91207036). There is no conflict of interest for
any of the authors. We thank Emmely de Vries, Merel Rijnsburger and Rianne van der Spek,
from the Department of Endocrinology & Metabolism, Academic Medical Center (AMC),
University of Amsterdam, Marja Neeleman from the Department of Clinical Chemistry,
Laboratory of Endocrinology, Academic Medical Center (AMC), University of Amsterdam,
and Simone Denis from the Laboratory Genetic Metabolic Diseases, Academic Medical
Center (AMC), University of Amsterdam, for experimental assistance.
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