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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

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trig

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rid

es

and

a p

aras

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ath

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erv

atio

n in

cre

ase

s p

lasm

a to

tal

cho

lest

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ese

Zu

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r ra

ts. (

A) G

rap

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ion

of s

tud

y d

esig

n (B

) Pla

sma

trig

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easu

rem

ents

dur

ing

the

exp

erim

ent (

* p

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com

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to fa

/fa

Sham

) (C

) VLD

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sec

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on

rate

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es a

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) Pla

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) Bo

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(F) A

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P <

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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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