ORIGINAL CONTRIBUTION

Differential effects of high-carbohydrate and high-fat dietson hepatic lipogenesis in rats

Alessandra Ferramosca • Annalea Conte •

Fabrizio Damiano • Luisa Siculella •

Vincenzo Zara

Received: 10 July 2013 / Accepted: 22 October 2013

� Springer-Verlag Berlin Heidelberg 2013

Abstract

Purpose Hepatic fatty acid synthesis is influenced by

several nutritional and hormonal factors. In this study, we

have investigated the effects of distinct experimental diets

enriched in carbohydrate or in fat on hepatic lipogenesis.

Methods Male Wistar rats were divided into four groups

and fed distinct experimental diets enriched in carbohy-

drates (70 % w/w) or in fat (20 and 35 % w/w). Activity

and expression of the mitochondrial citrate carrier and of

the cytosolic enzymes acetyl-CoA carboxylase and fatty

acid synthetase were analyzed through the study with

assessments at 0, 1, 2, 4, and 6 weeks. Liver lipids and

plasma levels of lipids, glucose, and insulin were assayed

in parallel.

Results Whereas the high-carbohydrate diet moderately

stimulated hepatic lipogenesis, a strong inhibition of this

anabolic pathway was found in animals fed high-fat diets.

This inhibition was time-dependent and concentration-

dependent. Moreover, whereas the high-carbohydrate diet

induced an increase in plasma triglycerides, the high-fat

diets determined an accumulation of triglycerides in liver.

An increase in the plasmatic levels of glucose and insulin

was observed in all cases.

Conclusions The excess of sucrose in the diet is con-

verted into fat that is distributed by bloodstream in the

organism in the form of circulating triglycerides. On the

other hand, a high amount of dietary fat caused a strong

inhibition of lipogenesis and a concomitant increase in the

level of hepatic lipids, thereby highlighting, in these con-

ditions, the role of liver as a reservoir of exogenous fat.

Keywords Fatty acid synthesis � Mitochondrial

citrate carrier � Lipogenic enzymes

Introduction

Hepatic lipogenesis is strictly regulated by a series of

hormonal and nutritional factors [1, 2]. In particular, the

effect of distinct nutrients and/or of different combinations

of them in experimental diets has been studied both in

animal models and in humans. It has been found that a diet

enriched in carbohydrate is capable of stimulating hepatic

fatty acid synthesis, thereby channelling the excess of

acetyl units deriving from glucose catabolism toward fat

biosynthesis in liver [1, 3]. On the contrary, a diet enriched

in fat generally decreases hepatic fatty acid synthesis even

if the final effect of this dietary treatment strongly depends

on the type and the amount of fat ingested [4–6]. In fact, a

diet enriched in polyunsaturated fatty acids (PUFA)

inhibits hepatic fatty acid synthesis more strongly than a

diet enriched in monounsaturated fatty acids (MUFA) or

saturated fatty acids (SFA) that, under certain conditions,

appear almost without effect. Furthermore, more fat is

ingested and higher inhibition of hepatic fatty acid syn-

thesis is found. All these effects imply the modulation of

several enzyme activities and are dependent on a differ-

ential gene expression induced by dietary fat.

The excess of carbon units introduced by hypercaloric

diets and mainly deriving from carbohydrates can be

addressed toward the anabolic pathway of hepatic fatty

acid synthesis. In this way, the excess of dietary energy is

stored as fat. However, the metabolic conversion of

A. Ferramosca � A. Conte � F. Damiano � L. Siculella �V. Zara (&)

Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali,

Universita del Salento, Via Provinciale Lecce-Monteroni,

73100 Lecce, Italy

e-mail: vincenzo.zara@unisalento.it

123

Eur J Nutr

DOI 10.1007/s00394-013-0613-8

carbohydrates into fatty acids involves a complex series of

reactions located in different subcellular compartments.

The mitochondrial acetyl-CoA deriving from catabolism is

at first condensed with oxaloacetate, thereby forming cit-

rate. This tricarboxylate is transported outside mitochon-

dria by the citrate carrier (CIC) [7, 8], an integral protein of

the inner mitochondrial membrane, and once in the cytosol

is again converted into acetyl-CoA and oxaloacetate by the

ATP citrate lyase. The cytosolic acetyl-CoA is then the

starter of hepatic fatty acid synthesis catalyzed by acetyl-

CoA carboxylase (ACC) and fatty acid synthetase (FAS).

In this anabolic pathway, an interesting role is played by

the CIC, which represents the only way to ensure that the

excess of carbon units produced in mitochondria is

addressed toward cytosolic biosynthesis. The CIC cata-

lyzes an electroneutral exchange of a tricarboxylate (cit-

rate, isocitrate, or cis-aconitate) plus a proton for a

dicarboxylate (malate or succinate) or phosphoenolpyr-

uvate, but the substrates transported and the direction of

fluxes across the inner mitochondrial membrane depend on

the metabolic state of the organism [9].

The information on the effect of various experimental

diets on hepatic fatty acid synthesis is extensive but, at the

same time, fragmentary and/or partially inconsistent. This

is mainly due to the very large variety of experimental

conditions used in the multitude of nutritional studies

carried out on this topic. We have therefore investigated

the effects of high-carbohydrate or high-fat diets on hepatic

lipogenesis using the same experimental conditions and the

same animal model. Moreover, besides the changes in the

activities and the expression of the cytosolic lipogenic

enzymes ACC and FAS, we have also investigated the

transport activity and the expression of the mitochondrial

CIC, a member of the mitochondrial carrier family [9, 10].

The picture emerging from this investigation shows not

only an elegant flexibility of hepatic fatty acid synthesis in

strict dependence on the experimental diets but also a

prompt and concerted modulation of mitochondrial and

cytosolic reactions.

Materials and methods

Animals and diets

Male Wistar rats (150–200 g) were obtained from Harlan

and housed individually in animal cages at a temperature of

22 ± 1 �C with a 12/12-h light–dark cycle and 30–40 %

humidity. After acclimatization, animals were divided into

four groups and fed with different diets for various time

periods, ranging from 1 to 6 weeks (Table 1). The first

group (control group) received a standard natural-ingredi-

ent diet, also known as standard rodent chow, containing

6.2 % (w/w) fat and 44.2 % (w/w) carbohydrate (5 % w/w

sucrose). The second group (HC group) received a diet

with 70 % (w/w) carbohydrate (66 % w/w sucrose) and an

almost equivalent amount of fat (5.2 % w/w). The third

group (HF group) was fed with a diet containing a higher

content of fat (20.2 % w/w fat) and a concomitant lower

amount of carbohydrate (45.5 % w/w). This diet was made

with a mixture of beef tallow plus hydrogenated fat, and as

a consequence, it contains a relatively high content of trans

fatty acids (17 % of total fatty acids). Approximate fatty

acid profile of this diet was as follows: 41 % saturated,

52 % monounsaturated, and 7 % polyunsaturated fatty

acids. The fourth group (HHF group) received a diet with

35.2 % (w/w) fat (exclusively lard) and 36.1 % (w/w)

carbohydrate. Approximate fatty acid profile of this diet

was as follows: 40 % saturated, 50 % monounsaturated,

and 10 % polyunsaturated fatty acids. Animals were

allowed ad libitum access to diet and water. At the

beginning of dietary treatment (week 0) and after 1, 2, 4,

and 6 weeks, rats were killed by decapitation, according to

the guidelines for the care and use of laboratory animals.

Body weight, liver weight, and food intake were recorded

throughout the study, ranging from 1 to 6 weeks of dietary

treatment. For the determination of plasma lipid, glucose,

and insulin, control and treated rats were starved overnight

before killing. Blood was collected and centrifuged to

separate plasma. Glucose concentration was determined

using a commercial kit. Plasma insulin concentration was

analyzed with an enzyme-linked immunosorbent assay kit.

Table 1 Composition of experimental diets

Control HC

diet

HF

diet

HHF

diet

Protein (g/100 g) 18.6 17.7 20.9 20.4

Fat (g/100 g) 6.2 5.2 20.2 35.2

Fatty acids (%)

SFA 16.0 15.0 41.0 40.0

MUFA 23.0 23.0 52.0 50.0

PUFA 61.0 62.0 7.0 10.0

Carbohydrate (g/100 g) of

which sucrose

44.2 70.0 45.5 36.1

5.0 66.0 18.2 15.0

kcal/g 3.1 4.0 4.5 5.4

Calories from protein (%) 24.0 17.8 18.7 15.0

Calories from fat (%) 18.0 11.8 40.6 58.4

Calories from carbohydrates

(%)

58.0 70.4 40.7 26.6

Control animals received a standard diet (Global Diet 2018S from

Harlan Teklad). The HC group received a diet with 70 % carbohy-

drate (Diet TD.98090 from Harlan Teklad), the HF group was fed

with an adjusted fat (20.2 % fat) diet (Diet TD.96132 from Harlan

Teklad), and the HHF group received a diet with 35.2 % fat (Diet

TD.03584 from Harlan Teklad). Energy content was calculated using

4 kcal/g for protein and carbohydrate, and 9 kcal/g for lipid

Eur J Nutr

123

Citrate transport in rat liver mitochondria

Rat liver mitochondria were prepared by standard pro-

cedures, and mitochondrial protein concentration was

determined by the Bradford method [11]. Freshly iso-

lated mitochondria were resuspended in 100 mM KCl,

20 mM Hepes, 1 mM EGTA, 2 lg/ml rotenone, pH 7.0,

at a concentration of about 5 mg protein/ml, and loaded

with L-malate. The assay of citrate transport, carried out

at 9 �C, was initiated by the addition of 0.5 mM

[14C]citrate and stopped by 12.5 mM 1,2,3-BTA. Rat

liver mitochondria were reisolated at 18,000 g for

10 min, washed, and then extracted with 20 % HClO4.

The mixture was centrifuged, and the radioactivity

present in the supernatant was counted by liquid

scintillation.

Reconstitution of the citrate transport into liposomes

Rat liver mitochondria (10–15 mg protein) were solubi-

lized with a buffer containing 3 % Triton X-100 (w/v),

20 mM Na2SO4, 1 mM EDTA, 10 mM Pipes, pH 7.0, at

a final concentration of about 10 mg protein/ml. After

incubation for 10 min on ice, the mixture was centri-

fuged at 25,000g for 20 min at 2 �C. About 600 ll of

the supernatant (mitochondrial extract) was supplemented

with 2 mg/ml cardiolipin, chromatographed onto a cold

hydroxyapatite column (pasteur pipette containing

600 mg of dry material), and eluted with a buffer con-

taining 0.5 % Triton X-100 and 5 mM citrate/NaOH, pH

7.0. The first 600 ll of the eluate was pooled and used

for the reconstitution experiments; protein concentration

was determined using the Lowry method modified for

the presence of Triton [12].

The composition of the initial mixture used for

reconstitution was as follows: 50 ll of hydroxyapatite

eluate, 90 ll of 10 % Triton X-114, 20 ll of 20 mg/ml

cardiolipin, 100 ll of 10 % phospholipids in the form of

sonicated liposomes, 70 ll of 100 mM Pipes (pH 7.0),

and 35 ll of 200 mM citrate in a final volume of 700 ll.

After vortexing, this mixture was passed 15 times

through the same Amberlite XAD-2 column, in order to

obtain the proteoliposomes. The external citrate was

removed by gel filtration on a Sephadex G-75 column.

The first 600 ll of the turbid eluate was collected, dis-

tributed in reaction vessels (180 ll), and used for the

transport studies. Transport at 25 �C was started by

adding 0.5 mM [14C]citrate (unless otherwise indicated)

to the proteoliposomes and terminated by the addition of

20 mM 1,2,3-BTA. The external radioactivity was

removed on Dowex AG1-X8 columns, and the internal

radioactivity was measured by scintillation counting.

Assay of lipogenic enzymes

The enzymatic activities of ACC and FAS were determined

spectrophotometrically in rat liver cytosol. Cytosol was

obtained by centrifuging the post-mitochondrial superna-

tant at 20,000g for 20 min at 2 �C. The pellet was dis-

carded, and the supernatant was then centrifuged at

105,000g for 60 min. The activities of ACC and FAS were

measured in the resulting cytosol as previously described,

using a NADH- and NADPH-linked assay, respectively

[13].

Western blot analysis

Polyacrylamide gel electrophoresis was performed in the

presence of 0.1 % SDS (SDS–PAGE) according to the

standard procedures. The mitochondrial proteins, separated

by SDS–PAGE, were then transferred to nitrocellulose

membrane. For protein detection, antisera prepared in our

laboratory and directed against the C-terminus of the rat

liver CIC and against the mammalian porin were used at a

dilution of 1:3 9 103. The immunoreacted proteins were

detected by the peroxidase reaction, using N, N0 Diamino

Benzydine (DAB) and hydrogen peroxide.

Isolation of RNA from rat liver and real-time qPCR

analysis

Total RNA extraction from rat liver was performed as

described in [14]. Quantitative gene expression analysis was

performed (SmartCycler System, Cepheid) using SYBR

Green technology (FluoCycle, Euroclone) and 18S rRNA for

normalization. The primers used for real-time PCR analysis

were the following: CICfor (50-GCCTCAGCTCCTTG

CTCTA-30); CICrev (50-ACTACCACTGCCTCTGCCA-30);FASfor (50-CTCTGGTGGTGTCTACATTTC-30); FASrev

(50-GAGCTCTTTCTGCAGGATAG-30); ACCFor (50-CTT

GGAGCAGAGAACCTTCG-30); ACCRev (50-CCTGG

ATGGTTCTTTGTCCC-30).

Assay of lipids

Total lipids were extracted from rat liver with a 1:1 mixture

of chloroform and methanol. The extracts were dried under

nitrogen flow and resuspended in a suitable volume of

0.1 % Triton X-100 before carrying out lipid analysis. The

individual assays of liver and plasma triglycerides, cho-

lesterol, and phospholipids were carried out using com-

mercial kits purchased from Futura System (Formello,

Italy; REF. N� 2703, 2049, 2302).

Eur J Nutr

123

Statistical analysis

GraphPad Prism� software version 5.0 (GraphPad Soft-

ware Inc., LA Jolla, CA, USA) was used for statistical

analyses. Data were confirmed to be normally distributed

by Anderson–Darling normality test (P [ 0.05). Values,

presented as the mean ± SD, were analyzed by one-way

ANOVA. A Tukey–Kramer post hoc analysis was used to

detect significant differences between the means at a level

of P \ 0.05.

Results

Food intake, body weight, and liver weight

As illustrated in Table 1, the increase in dietary fat was

compensated by a reduction in carbohydrate levels,

whereas the protein concentration was kept approximately

constant (around 20 % w/w) in all diets. Because of the

different caloric values of carbohydrate and lipid, the diets

slightly differed in this parameter, being 3.1, 4.0, 4.5, and

5.4 kcal/g for control, HC, HF, and HFF diet, respectively.

Furthermore, the approximate fatty acid profile (SFA,

MUFA, and PUFA) of both high-fat diets (HF and HFF)

was comparable and low in PUFA in order to prevent a

strong inhibitory effect of hepatic fatty acid synthesis by

these unsaturated fatty acids.

As shown in Table 2, food intake was approximately the

same in control, HC-, and HF-fed rats, whereas a small

decrease was seen in the case of the HFF-fed animals.

Because of the different caloric value of the diets, the kcal

introduced per day slightly increased passing from control

to HC-, HF-, and HFF-fed animals. However, these dif-

ferences were not statistically significant. With the excep-

tion of the first week, the body weight of the HC-fed rats

was practically similar to that of the control group

(Table 2). In the HF group, the body weight was signifi-

cantly higher in comparison with that of control animals in

the first two weeks of dietary administration. The HHF

animals showed a body weight significantly higher than

that of control rats at any time of dietary treatment. The

maximal increase in body weight in HHF animals was

around 20 % with respect to control rats (Table 2). Finally,

no statistically significant variations were found in the liver

weight of animals fed with the different experimental diets

(Table 2).

Hepatic fatty acid synthesis

Figure 1a shows the CIC transport activity measured in

freshly isolated liver mitochondria from rats fed the four

experimental diets. A small increase in the activity of

mitochondrial CIC (about 15–20 %) was found in HC-fed

animals when compared to control rats. This increase was

statistically significant at the fourth and the sixth week of

Table 2 Effect of dietary treatments on food intake, body weight, and liver weight

Food intake

Control HC HF HHF

Mean SD Mean SD Mean SD Mean SD

g/day 20.5 2.8 19.3 5.0 18.8 2.4 16.0 4.6

kcal/day 63.5 8.7 77.2 20.0 84.4 9.7 86.2 18.6

Body weight (g)

Week 0 234.0 7.8 – – –

Week 1 232.5 3.5 254.0* 14.1 281.3*� 15.7 282.3*� 10.5

Week 2 240.0 17.0 236.2 24.9 265.7*� 15.5 282.7*�� 6.2

Week 4 296.3 0.6 289.2 29.3 308.0 38.9 302.0* 22.2

Week 6 281.7 13.9 316.0 50.8 330.7 46.5 305.7* 21.6

Liver weight (g/100 g body weight)

Week 0 3.28 0.26 – – –

Week 1 3.85 0.57 4.18 0.61 3.70 0.91 3.58 0.57

Week 2 3.80 0.47 3.74 0.80 3.20 0.17 3.02 0.64

Week 4 3.90 0.63 3.53 0.70 3.15 0.57 3.01 0.40

Week 6 3.47 0.30 3.25 0.47 3.08 0.55 2.85 0.52

Food intake, body weight, and liver weight of rats fed with the experimental diets used in this study are shown for the times indicated. Each point

represents the mean ± SD for 4 animals

* P \ 0.05 versus rats fed a control diet; �P \ 0.05 versus rats fed a HC diet; �P \ 0.05 versus rats fed a HF diet

Eur J Nutr

123

dietary treatment. On the contrary, a statistically significant

decrease in the CIC activity was observed in HF animals at

the beginning of dietary treatment in comparison with both

control and HC group. The highest CIC inhibition in the

HF-fed rats was found at the first week (52.3 and 57.4 %

vs. control and HC, respectively). Interestingly, the HHF

group showed a higher CIC activity inhibition in compar-

ison with the HF group at the first week. In fact, at that

time, the CIC inhibition found in the HHF animals was

80.4 and 82.5 % in comparison with the control and the HC

group, respectively. It is noteworthy that the mitochondrial

CIC activity became indistinguishable in HF, HHF, and

control animal at longer dietary treatments (Fig. 1a, sixth

week).

To exclude a possible influence of the diet on the lipid

composition of the inner mitochondrial membrane in which

CIC operates, we purified the carrier protein and recon-

stituted its transport activity into liposomes. As shown in

Fig. 1b, the behavior of the CIC activity in the proteolip-

osomal system, which shows a definite lipid composition,

was very similar to that previously observed in intact rat

liver mitochondria. Very interestingly, at the first week of

dietary treatment, we found an inhibition of the CIC

transport activity higher than 80 % in HHF animals with

respect to both the control and the HC ones. It is also

remarkable the progressive quenching of the differences in

CIC activity over time. At the sixth week of dietary

treatment, the CIC activities were comparable in control,

HF, and HHF animals (Fig. 1b). Only a small yet signifi-

cant increase was still detectable in HC-fed rats in com-

parison with the other experimental groups.

Table 3 reports the analysis of the kinetic parameters

(Km and Vmax) of mitochondrial CIC activity in the

reconstituted system. No change in the affinity of the CIC

toward its substrate was detected at any time of dietary

treatment in any animal group. On the contrary, the vari-

ations in the Vmax values were consistent with the CIC

activities measured both in fresh mitochondria and in the

reconstituted system (Fig. 1a, b). Indeed, a small increase

in the Vmax of CIC was found in HC-fed rats in com-

parison with the control animals at any time of dietary

treatment. On the contrary, a strong decrease in the Vmax

was found in HF and HHF animals during the first 2 weeks

of dietary treatment.

Figure 2 shows a Western blot analysis of the mito-

chondrial membranes purified from the four groups of

animals and immunodecorated with an antiserum directed

against the rat liver CIC. The variations in the expression

of the mitochondrial CIC in each group of rats (Fig. 2a)

were consistent with those found in the CIC transport

activity previously shown in Fig. 1a, b. The analysis of the

CIC transcripts showed similar trends (Fig. 2b).

Figure 3a shows that the ACC activity slightly and

significantly increased in the HC-fed rats in comparison

with the control animals at any time of dietary treatment.

The extent of this increase (about 15–20 %) was compa-

rable to that found in the CIC activity in the same group of

animals. On the contrary, a strong and significant inhibition

was found in the HF- and HHF-fed rats at the first week of

dietary treatment in comparison with both control and HC-

fed rats (Fig. 3a). Again, this inhibition was progressively

reversed over time and nullified at the sixth week. As

expected, the FAS activity showed a behavior very similar

to that of ACC (Fig. 3b). Notably, the maximal inhibition

of ACC and FAS activity in HHF-fed animals in compar-

ison with the control ones was 77.5 and 74.3 %, respec-

tively. The decrease in the ACC and FAS activities was

further substantiated by the assay of AAC and FAS

mRNAs as shown in Fig. 3c, d, respectively.

MitochondriaA

Proteoliposomes

Control

B

Cit

rate

up

take

(nm

ol .

min

-1 . m

g p

rote

in-1

)C

itra

te u

pta

ke(n

mo

l . 1

0 m

in-1

. m

g p

rote

in-1

)

3

6

9

12

15

18

0 1 2 3 4 5 6

Weeks

HC

HF

HHF

50

100

150

200

250

300

350

0 1 2 3 4 5 6

Weeks

Control

HC

HF

HHF

Fig. 1 Effect of high-carbohydrate and high-fat diets on the transport

activity of mitochondrial CIC. Transport of citrate into rat liver

mitochondria (a) and into proteoliposomes (b) was measured at the

times indicated. The values reported in the figure represent the

mean ± SD (n = 4). *P \ 0.05 versus rats fed a control diet;�P \ 0.05 versus rats fed a HC diet; �P \ 0.05 versus rats fed a HF

diet

Eur J Nutr

123

Plasma and liver lipids

Table 4 shows that plasma triglycerides strongly and sig-

nificantly increased in HC-fed rats in comparison with the

control animals. The highest increment in plasma triglyc-

erides was detected in the HC group at the first week

(?2.3-fold vs. control group). A moderate, yet significant,

increment in plasma triglyceride levels was also found in

HF-fed rats in comparison with the control animals.

Notably, the HHF-fed rats showed a high peak in the tri-

glyceride levels at the first week of dietary treatment

(?2.1-fold vs. control group), followed by lower values at

longer feeding times (Table 4). The assay of plasma cho-

lesterol did not reveal any significant variation among the

different dietary groups except for a small yet significant

increase in the cholesterol level in the HHF-fed animals at

the first week of dietary treatment (?26.8 % with respect to

control) (data not shown). In the case of plasma phospho-

lipids (data not shown), statistically significant variations

were found only in the first week of dietary treatment. The

following increments were indeed found: ?34.8 % (HC

group), ?57.2 % (HF group), and ?75.3 % (HHF group)

with respect to control animals.

Figure 4a shows that the triglyceride levels strongly

increased in liver of HHF-fed rats in comparison with the

control rats at any time of dietary treatment. In particular, the

highest increments (around ?2.5-fold) were found starting

from the second week up to the sixth week of dietary

administration. A moderate increase in the liver triglyceride

levels was also found in HF-fed rats in comparison with the

control animals at the fourth week (?38 %), whereas a

strong increase was found at the sixth week (?2.3-fold). On

the contrary, the HC animals showed modest, although sig-

nificant, variations in the liver triglyceride concentrations

only in the first two weeks of dietary treatments. A compa-

rable behavior was observed in the case of the liver

cholesterol levels (Fig. 4b). In fact, the cholesterol concen-

trations more than doubled in HHF-fed animals in compar-

ison with the control rats starting from the second week of

dietary administration. Moreover, also in the HF-fed ani-

mals, a significant increase in the liver cholesterol levels was

reproducibly found at the fourth and the sixth week. On the

contrary, in HC-fed animals, a significant increase in the

levels of liver cholesterol was only found in the first 2 weeks

of dietary treatment (Fig. 4b). No significant variations were

detected in the levels of liver phospholipids in any animal

group (data not shown).

Plasma levels of glucose and insulin

Figure 5a shows that the plasma glucose levels doubled in

HC-fed rats in comparison with the control animals.

Moreover, in the HF- and HHF-fed animals, a significant

increase in the glucose levels was found at any time of

dietary treatment in comparison with the control rats.

However, these increments were lower than those found in

the HC-fed animals. Only at the sixth week, the glucose

levels in the HC-, HF-, and HHF-fed animals were indis-

tinguishable (about 60 % higher than the values found in

control animals). Notably, the variations in the plasma

insulin levels (Fig. 5b) very closely reflected those in

glucose levels (Fig. 5a).

Discussion

The de novo fatty acid synthesis is a metabolic process

actively investigated because its regulation is quite com-

plex and also because its dysregulation may lead to several

diseases. In order to gain more insights into this anabolic

process, we have investigated the effect of three different

diets on rat hepatic lipogenesis.

Table 3 Km and Vmax of citrate transport in a reconstituted system

Km (mM) Vmax (nmol�min-1�mg protein-1)

Control HC HF HHF Control HC HF HHF

Week 0 0.198 – – – 141.73 – – –

Week 1 0.189 0.189 0.190 0.198 143.26 164.75 72.06 32.62

Week 2 0.203 0.211 0.193 0.187 140.96 158.20 77.64 78.51

Week 4 0.203 0.213 0.183 0.184 140.96 159.36 113.40 112.17

Week 6 0.189 0.196 0.187 0.195 141.59 168.07 141.55 145.46

Km and Vmax values were measured in a reconstituted system at the times indicated. Proteoliposomes were reconstituted with the CIC as

described in the ‘‘Methods’’ section. [l4C]Citrate, 0.04–0.40 mM, was added to proteoliposomes containing 10 mM citrate. The citrate/citrate

exchange was stopped 1 min after the addition of the radiolabeled substrate by 20 mM 1,2,3-BTA. Km and Vmax values were calculated by

linear regression

Data correspond to the mean of values obtained in two independent experiments. Differences between values of every couple were less than

10 %

Eur J Nutr

123

Some methodological aspects of this study merit con-

sideration before discussing in details the results obtained.

At first, the feeding treatment of rats with distinct diets,

enriched in sucrose or in fat, has been done in the same

study. This helped us to investigate the differential effects

of carbohydrate, on the one hand, and of fat, on the other

hand, in the same experimental conditions. On the contrary,

general conclusions on diet-induced metabolic changes

have been reached by combining the results obtained in

independent studies, carried out in different experimental

conditions [3, 5, 15, 16]. Secondly, in this study, we

investigated the changes occurring in rat hepatic lipogen-

esis in a feeding period ranging from 1 to 6 weeks, thereby

highlighting the impressive changes of this anabolic path-

way especially in the first week. Third, we have analyzed

the molecular mechanisms of these metabolic changes

involving a strict coordination between mitochondrial and

cytosolic reactions, thus clearly demonstrating a strict

interplay between anabolic and catabolic pathways.

An interesting consideration can be made on body

weight changes. Although the differences were not signif-

icant, there is a clear trend in this study showing that when

the energy content of the diet is higher, the intake of food

becomes lower (Table 2). Although the four groups of

animals ate a similar number of calories, the gains in body

weight were different and the effect is especially evident

for HF and HHF diets, which seem to show a higher

feeding efficiency (weight gain per kcal consumed). In this

case, the reported effects of the increase in dietary fat on

the secretion and signaling of gastrointestinal hormones

may predispose to weight gain and obesity [17].

The enzymatic assays showed the elevated metabolic

flexibility of liver, which was able to quickly modulate the

de novo fatty acid synthesis in relation to the type of diet

ingested. A diet containing 66 % (w/w) sucrose moderately

stimulated hepatic lipogenesis (about ?20 % with respect

to control animals). Similar results were previously

reported in the literature, although a more prominent

increase in hepatic lipogenesis was detected in the first

hours up to a few days after the ingestion of a HC-enriched

diet [15]. However, in most of these studies, the animals

were either first starved for some time [3, 15, 18] or fed

with a high-fat and carbohydrate-free diet [1] before

receiving the HC diet. The HC diet, on the other hand,

contained a low level of fat [3]. All these experimental

expedients significantly emphasized the metabolic changes

due to a HC diet. However, these enzyme hyperinductions,

although important for experimental purposes, have a

scarce significance in the current nutritional models of

western populations. Indeed, the current western dietary

habits generally imply an enrichment in the sucrose of diets

that already contain adequate (or sometimes excessive)

amounts of fat and protein with the consequence of

ingestion of unbalanced diets and/or of excess of calories

[19]. The dietary manipulations reported in our investiga-

tion are more similar to these eating behaviors.

Plasmatic lipid profile of rats fed the HC diet suggested

interesting considerations. As shown in Table 4, in these

animals, the plasma triglyceride levels were significantly

higher than those of control rats in the first 4 weeks of

feeding. A concomitant increase in hepatic lipogenesis and

plasma triglycerides is therefore evident in HC-fed rats.

The increase in the activity of mitochondrial CIC and

lipogenic enzymes ACC and FAS stimulated the synthesis

of fatty acids in liver with a subsequent rise in the level of

plasma triglycerides, as already demonstrated by others

A

B

Fig. 2 Effect of high-carbohydrate and high-fat diets on protein

levels (a) and mRNA (b) of the mitochondrial CIC. The values

reported in the graph represent the mean ± SD (n = 4; *P \ 0.05 vs.

rats fed a control diet; �P \ 0.05 vs. rats fed a HC diet; �P \ 0.05 vs.

rats fed a HF diet). The amount of CIC revealed by immunodeco-

ration and the amount of CIC mRNA at the beginning of dietary

treatment were set to 100 %. The values reported in the graph

represent the mean ± SD (n = 3; *P \ 0.05 vs. rats fed a control

diet; �P \ 0.05 vs. rats fed a HC diet; �P \ 0.05 vs. rats fed a HF diet)

Eur J Nutr

123

[15, 20, 21]. The increase in plasma triglycerides observed

in the present investigation was persistent in the first month

of feeding treatment, thereby substantiating the role of liver

in synthesizing and subsequently distributing lipids to other

tissues by circulation. On the other hand, in our study, no

statistically significant variations in the level of liver lipids

A B

C D

Fig. 3 Effect of high-carbohydrate and high-fat diets on lipogenic

enzyme activities and mRNA levels. The activities of ACC (a) and

FAS (b) were measured in the cytosol of rat hepatocytes at the times

indicated. The values are expressed as nanomoles of NADH (ACC) or

NADPH (FAS) oxidized�min-1�mg protein-1 and represent the

mean ± SD (n = 4; *P \ 0.05 vs. rats fed a control diet;�P \ 0.05 vs. rats fed a HC diet; �P \ 0.05 vs. rats fed a HF diet).

Total RNA from the liver of rats fed the experimental diets was

extracted, and the abundance of ACC (c) and FAS (d) mRNA was

analyzed by RT-quantitative PCR. The amount of ACC and FAS

mRNA revealed at the beginning of dietary treatment was set to

100 %. The values reported in the graph represent the mean ± SD

(n = 3; *P \ 0.05 vs. rats fed a control diet; �P \ 0.05 vs. rats fed a

HC diet; �P \ 0.05 vs. rats fed a HF diet)

Table 4 Plasma triglycerides (mg/dl)

Control HC HF HHF

Mean SD Mean SD Mean SD Mean SD

Week 0 119.0 11.2 – – –

Week 1 125.5 23.0 288.0* 11.6 162.7*� 6.3 260.7*�� 5.4

Week 2 156.3 8.1 260.1* 4.5 171.5� 18.8 154.4� 12.5

Week 4 146.2 8.1 293.0* 43.9 231.6*� 26.8 157.6�� 4.5

Week 6 157.6 13.4 192.4* 26.8 199.4* 8.1 206.3* 1.8

The levels of plasma triglycerides were determined at the times indicated, using commercial kits. The values reported in the table represent the

mean ± SD (n = 4)

* P \ 0.05 versus rats fed a control diet; � P \ 0.05 versus rats fed a HC diet; � P \ 0.05 versus rats fed a HF diet

Eur J Nutr

123

have been found in HC-fed rats. Indeed, only a moderate,

but statistically significant, increase in triglyceride and

cholesterol levels was found in the first 2 weeks of HC diet

feeding.

A strong inhibition of lipogenic enzyme expression and

activity was instead found in rats fed with a diet enriched in

fat. Two fat-rich diets were used, one containing 20 % (w/w)

and the other 35 % (w/w) fat. The de novo fatty acid syn-

thesis inhibition induced by these experimental diets was

particularly strong in the first week of dietary treatment. This

inhibition progressively decreased over time and was com-

pletely abolished at the sixth week of dietary treatment. A

similar time dependence of lipogenesis inhibition was

recently found in another study [22]. Furthermore, we found

that the degree of inhibition was also dependent on the

amount of dietary fat (about 80–90 and 50–55 % inhibition at

35 and 20 % (w/w) dietary fat, respectively). By further

lowering the total level of dietary fat, only diets enriched in

PUFA were able to significantly reduce hepatic lipogenesis

[6, 23–27]. The PUFA-mediated inhibition of hepatic lipo-

genesis was also observed when rats were fed, over the long

term, with a high-fat (45 % of calories) diet. In these animals,

a decrease in the amount of mRNA coding for hepatic lipo-

genic enzymes was found in comparison with the rats fed

with an isocaloric diet rich in saturated fatty acids [28]. On

the other hand, it has been shown that an excessive level of

dietary fat counteracts the differential effect of the various

types of fatty acids on hepatic lipogenesis, thereby resulting

in a massive inhibition of hepatic lipid metabolism [16].

Therefore, the effects of dietary fat on hepatic lipogenesis

were dependent on a series of factors, not only the amount of

fat in the diet, but also the fatty acid composition, the length,

and the characteristics of feeding treatment (i.e., feeding

ad libitum or fasting and refeeding) and the animal species

tested [4, 5, 29].

A

B

Fig. 4 Effect of high-carbohydrate and high-fat diets on liver lipids.

The levels of liver triglycerides (a) and cholesterol (b) were

determined at the times indicated. Each point represents the

mean ± SD for 4 liver samples. *P \ 0.05 versus rats fed a control

diet; �P \ 0.05 versus rats fed a HC diet; �P \ 0.05 versus rats fed a

HF diet

A

B

Fig. 5 Effect of high-carbohydrate and high-fat diets on plasmatic

levels of glucose and insulin. a Plasma glucose concentrations were

determined using a commercial kit. b Plasma insulin concentrations

were analyzed with commercial enzyme-linked immunosorbent assay

kits. The values reported in the figure represent the mean ± SD

(n = 4). *P \ 0.05 versus rats fed a control diet; �P \ 0.05 versus

rats fed a HC diet; �P \ 0.05 versus rats fed a HF diet

Eur J Nutr

123

Notably, in the present study, parallel inhibitions of

mitochondrial CIC and cytosolic ACC and FAS were

found, thus suggesting a concerted network of regulation

that starts from substrate exit from mitochondria and

propagate to cytosolic anabolic reactions. This type of

concerted inhibition of mitochondrial and cytosolic reac-

tions was also found in starved rats [30]. The intricacy of

these regulations is also corroborated by the fact that ACC

plays a crucial role in the de novo lipogenesis because its

product, malonyl-CoA, is not only an intermediate of fatty

acid synthesis but also an inhibitor of carnitine palmitoyl-

transferase (CPT-1) and therefore an inhibitor of fatty acid

oxidation [31]. Moreover, plasma triglycerides more than

doubled in HHF animals at the first week of feeding but

after that decreased to control values at the second week

(Table 4).

The high level of circulating triglycerides found only at

the first week in the HHF group cannot be due to an

increased de novo fatty acid synthesis because the activities

of both mitochondrial CIC and ACC/FAS were profoundly

depressed in these animals at that time (Figs. 1, 3).

Therefore, these triglycerides are of exogenous origin,

most probably deriving from the high fat content of the diet

administered to these animals. On the other hand, it is

particularly evident that the extremely high amount of

triglycerides was found in liver, starting from the second

week of feeding. In principle, this elevated level of lipid in

liver, reported in this and in other studies [22, 32–34], can

be caused by several factors, such as an increased uptake

and esterification of fatty acids from dietary sources, an

increased de novo fatty acid synthesis and a decreased fatty

acid oxidation. The strong inhibition of hepatic lipogenesis

occurring in HHF-fed rats (Figs. 1, 2, 3) suggests that liver

may represent an energy storage for fat of exogenous ori-

gin. It is also surprising to observe in HHF animals that

when plasma triglycerides are high (first week), liver tri-

glycerides are low, whereas the opposite happens at longer

times of feeding, that is, a great accumulation of fat in liver

and a normalization of triglyceride levels in plasma. Fur-

thermore, in these animals, the same trend is observed for

cholesterol, that is, a moderate yet significant increase

(P \ 0.05 vs. rats fed a control diet) in its level in plasma

at the first week, followed by a normalization at longer

times. On the contrary, a specular trend was observed in

TRIGLYCERIDES

CHOLESTEROL

CIC

Citrate

Acetyl-CoAOAA

Palmitic acid

ACC

FAS

Citrate

Glucose

Pyruvate

Pyruvate

OAAAcetyl-CoA +

Liver mitochondria

LIPOGENESISLIPOGENESIS

GLUCOSE

INSULIN

TRIGLYCERIDES

HC diet HF diet

Liver

BloodFig. 6 Metabolic pathways

influenced by high carbohydrate

and high-fat diets. Metabolic

changes occurring in rats under

the different nutritional

conditions used in this study.

OAA oxaloacetate

Eur J Nutr

123

liver, where only from the second week onward, an accu-

mulation of hepatic cholesterol was clearly observed.

Actually, the metabolic changes occurring in liver may be

more complicated than those described. In fact, the

increase in liver triglycerides starting from the second

week, most probably due to an accumulation of exogenous

lipids of dietary origin, can also derive, at least in part,

from a progressive reversal of lipogenesis inhibition.

An increase in the plasmatic levels of glucose and

insulin was observed in all the feeding conditions used

in this study, even if the highest increase was seen in the

case of the HC diet, especially in the first month of

treatment. The parallel increase in glucose and insulin in

the case of HC-fed rats is easily explained by the ele-

vated content of sucrose of this type of diet. On the

other hand, the high plasmatic level of glucose and

insulin in HF- and HHF-fed animals can be due to a

lower utilization of glucose when diets are strongly

enriched in fat, suggesting a condition of insulin resis-

tance [35, 36]. An excess of energy supplied by fat

corresponds to a scarce tissutal utilization of glucose,

thereby causing hyperglycemia and hyperinsulinemia.

Moreover, it has also been reported that an excess of

circulating free fatty acids, whose concentration was

significantly greater in animals fed a HF or a HC diet

[37], may contribute to insulin resistance [38].

Conclusions

Figure 6 illustrates the metabolic changes occurring in

liver of rats under the different nutritional conditions

used in this study. The changes occurring in the level of

circulating lipids are also illustrated. A diet enriched in

sucrose, but containing adequate amounts of protein and

fat, moderately stimulates hepatic lipogenesis. Modest

and transient increments in the levels of triglycerides and

cholesterol were observed in liver, whereas a strong

increase in triglycerides was found in plasma. This

suggests that the excess of sucrose is converted into fat

and that this fat (i.e., circulating triglycerides) is dis-

tributed by bloodstream in the organism. On the con-

trary, a diet enriched in fat, but containing adequate

amounts of carbohydrate and protein, caused a strong

inhibition of lipogenesis, especially at the beginning of

this dietary treatment (first 2 weeks). A time-dependent

reversal of this inhibition was then progressively found.

Starting from the second week of feeding, a prominent

increase in the level of hepatic triglycerides and cho-

lesterol was observed. Interestingly, a rapid increase in

the levels of circulating lipids was only found at the first

week of dietary treatment. Liver, therefore, appears as a

compensating reservoir of exogenous lipids in conditions

of inhibited de novo lipogenesis.

The overall picture emerging from this investigation

shows a prompt metabolic flexibility of hepatic fatty acid

synthesis in the dependence of the characteristics of the

diet and, in particular, of the amount and quality of the

nutrients introduced.

Conflict of interest None.

Ethics statement This study was carried out in strict accordance

with the European Committee Council 106 Directive (86/609/EEC)

and with the Italian animal welfare legislation (art 4 and 5 of D.L.

116/92). The Italian Ministry of Health specifically approved this

study.

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