University of Groningen

The role of apolipoprotein E in the assembly and secretion of very low density lipoproteinsMensenkamp, Arjen Rutger

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63

CHAPTER 3:

High carbohydrate intake stimulates very low density lipoprotein triglyceride

secretion without induction of lipogenic genes and hepatic steatosis

in C57BL/6J wildtype and apolipoprotein E-deficient mice

Arjen R. Mensenkamp1, Bas Teusink2, Rick Havinga1, Vincent W. Bloks1,

Julius F. W. Baller1, Louis M. Havekes2,3, Folkert Kuipers1

Groningen University Institute for Drug Exploration, 1Center for Liver, Digestive and

Metabolic Diseases, Faculty of Medical Sciences and University Hospital Groningen, Groningen, The Netherlands, 2Gaubius Laboratory TNO-PG, Leiden, The Netherlands,

Departments of 3Human Genetics and Cardiology, Leiden University, Leiden, The Netherlands.

Submitted

Chapter 3: Dietary carbohydrates and VLDL secretion

64

ABSTRACT

High carbohydrate diets stimulate hepatic VLDL secretion in humans and rodents. This has been attributed to induction of de novo lipogenesis. We have evaluated the effects

of high carbohydrate diets in C57BL/6J wildtype mice and in apolipoprotein (apo) E-deficient mice, which accumulate triglycerides (TG) in their livers and show impaired very low density lipoprotein-triglyceride (VLDL-TG) secretion when fed a normal chow

diet. Plasma TG and cholesterol levels were not affected in wildtype and apoE-deficient mice fed a high sucrose (40%) diet for two weeks compared to chow-fed animals, but free fatty acid levels were decreased by sucrose in both strains. Neither wildtype nor apoE-deficient mice fed the high sucrose diet showed increases in hepatic lipid contents.

Expression of genes encoding lipogenic enzymes, i.e., fatty acid synthase (Fas) and acylCoA carboxylase (Acc) were unchanged or even modestly decreased. VLDL-TG

secretion, however, was strongly increased in both wildtype (165.3 ± 21.4 vs 345.9 ±

29.0 µmol/kg/h) and apoE-deficient mice (92.9 ± 12.1 vs 217.4 ± 42.3 µmol/kg/h) by

sucrose. VLDL composition and size did not change. The increase in VLDL-TG secretion was associated with markedly increased protein levels of the microsomal triglyceride transfer protein (MTP). Expression of the Mttp gene and other genes involved in VLDL

secretion, i.e., Apob and diacylglycerol acyltransferase (Dgat) were modestly increased. Similar effects (i.e., increased VLDL-TG secretion, no change in hepatic fat content) were

observed after addition of 10% fructose to drinking water of mice for two weeks. We conclude therefore that dietary carbohydrate-induced VLDL-TG secretion in C57BL/6J mice occurs independently from induction of de novo lipogenesis and hepatic lipid

accumulation. The impairment of VLDL-TG secretion associated with apoE-deficiency is maintained under these dietary conditions.

The role of apoE in the assembly and secretion of VLDL

65

INTRODUCTION

High intake of dietary carbohydrates has been reported to stimulate de novo lipogenesis

and very low density lipoprotein-triglyceride (VLDL-TG) secretion in rats (1-5), mice (6,7), and humans (8,9). Hepatic steatosis is induced in adult rodents when fed a carbohydrate-rich diet continuously (1,3,4) and after fasting followed by short-term refeeding (7). The development of a fatty liver has been attributed to increased de novo lipogenesis by

induction of “lipogenic genes”, such as fatty acid synthase (Fas). Expression of Fas is regulated by the transcription factor sterol-regulatory element-binding protein-1c (SREBP-1c) (10), which is, in turn, controlled by insulin (7,11). The direct link between

lipogenesis and VLDL secretion is not yet clear: only a relatively small fraction of VLDL-TG appears to be derived from de novo synthesized fatty acids (12). There is growing interest in the metabolic actions of dietary carbohydrates as intake of a high-

carbohydrate diet in humans is associated with elevated plasma TG levels, which constitute an independent risk factor for development of atherosclerosis (8).

Apolipoprotein E (apoE) is a major constituent of all circulating lipoproteins, with the exception of low-density lipoproteins (LDL). Its best known function concerns its role

in LDL-receptor- and LDL-receptor-related protein (LRP)-mediated clearance of lipoproteins. Thus, apoE-deficiency in mice leads to elevated plasma cholesterol concentrations due to impaired clearance of remnant lipoproteins (13-15). In addition, these mice develop fatty livers when fed normal chow (16) and show an impaired VLDL-

TG secretion (15). These effects of apoE-deficiency are normalized upon introduction of the human APOE3 gene into the liver of Apoe-/- mice (17). The exact mechanism how apoE influences VLDL-TG secretion is not yet clear.

As apoE-deficient mice display strongly impaired hepatic VLDL-TG secretion and develop a fatty liver, the question was addressed whether or not high intake of dietary carbohydrates would have differential effects in these mice compared to C57BL/6J wildtype mice. There are data that indicate that expression of Apoe is induced in rodent

liver upon feeding high carbohydrate diets (18). Therefore, we fed C57BL/6J control and apoE-deficient mice with a normal rodent chow diet or with a high carbohydrate diet for two weeks. Our data show that feeding wildtype and apoE-deficient mice with a high

carbohydrate diet results in a strong induction of VLDL-TG secretion and protein levels of microsomal triglyceride transfer protein (MTP), without affecting expression of the genes controlling lipogenesis and accumulation of additional fat in the liver. However, VLDL-TG secretion in apoE-deficient mice remains significantly impaired when compared

to wildtype mice. These results show that, in C57BL/6J mice, carbohydrate-induced stimulation of VLDL-TG secretion occurs independently from induction of de novo lipogenesis and the presence of apoE.

Chapter 3: Dietary carbohydrates and VLDL secretion

66

MATERIALS AND METHODS

ANIMALS

The apoE-deficient (Apoe-/-) mice used for these studies have been described before (19).

C57BL/6J (wildtype) and Apoe-/- mice were housed in a light- and temperature-controlled environment. Male mice were used throughout the study at three to four months of age.

The animals received humane care and experimental protocols complied with local guidelines for use of experimental animals. DIET

Mice were fed low fat/low cholesterol lab chow containing 6.2% fat and approximately 0.01% cholesterol (wt/wt) (RMH-B, Hope Farms BV, Woerden, The Netherlands) or a semi-synthetic diet containing 40% sucrose, 40% starch, 16% casein, and 4% vitamins without fat or cholesterol (Hope Farms BV). Where indicated, separate groups of mice

received the chow diet and tap water supplemented with 10% fructose (20). Food and water were available ad libitum. PLASMA AND LIVER TISSUE SAMPLING

Mice were anaesthetized with halothane after a 9 h fast. A large blood sample for determination of plasma lipids and for VLDL isolation was collected by cardiac puncture. Subsequently, the liver was quickly removed, weighed and frozen in separate portions for RNA isolation and lipid analysis, respectively.

PLASMA LIPIDS AND INSULIN LEVELS

Plasma TG, free cholesterol, fatty acids and total cholesterol concentration were

determined using commercially available kits (Roche Diagnostics, Basel, Switzerland). Insulin levels were measured using a radio immuno assay (RI-13K, Linco Research Inc, St. Charles, MO).

IN VIVO VLDL-TG PRODUCTION RATE

The hepatic VLDL-TG production rate in control and Apoe-/- mice on different dietary regimes (n = 5 in all groups) was determined as described previously (15,17). In short,

mice were fasted 9 hours prior to the experiment and 12.5 mg Triton WR 1339 in 100 µl PBS was injected via the penile vein. Tail blood samples were taken under light halothane anesthesia before and at 1, 2, and 3 hours after injection of Triton WR 1339 for TG measurements. VLDL-TG production rate was calculated from the slope of the TG

concentration vs time curve.

The role of apoE in the assembly and secretion of VLDL

67

VLDL ISOLATION AND COMPOSITION

VLDL/IDL (d<1.019 g/ml) from fasted mice (9 h) was isolated as described previously (17,21). Plasma (350-400 µl) obtained before and four hours after injection of Triton WR 1339 was adjusted to 1 ml with a NaCl/NaBr solution with a density of 1.019 g/ml,

containing 1 mM EDTA and NaN3, and centrifuged at 120 000 rpm in a Beckman

Optima TM 102.2 rotor for 100 minutes at 4 °C (22). VLDL was isolated by tube slicing

and the volume was recorded by weight. TG and cholesterol content were determined as described. Phospholipids were determined using a commercially available kit (WAKO

Chemicals, Neuss, Germany). The difference between VLDL composition four hours after Triton WR 1339 injection and steady-state levels were used to estimate the composition of newly secreted nascent VLDL as described (23). VLDL SIZE DETERMINATION

VLDL size and size distribution were analyzed by dynamic light scattering using a Nicomp model 370 submicron particle analyzer (Nicomp Particle Sizing Systems, Santa Barbara, CA). Diameters were obtained from the volume distribution provided by the

analyzer. HEPATIC LIPID ANALYSIS

Livers were homogenized in buffer containing 50 mM tris (pH 7.4), 250 mM sucrose, 1

mM EDTA, 5 µg/ml leupeptin, 1 mM benzamidine, and 1 mM PMSF. Hepatic concentrations of TG, free and total cholesterol were measured using commercial kits (Roche Diagnostics) after lipid extraction according to Bligh and Dyer (24) and resolving the lipid in 2% Triton X-100 in water. Phospholipid content of liver tissue was

determined according to Böttcher et al. (25) after lipid extraction. HEPATIC RNA ISOLATION

Total mRNA was isolated from 30 mg liver tissue with the Trizol method (GIBCO, Breda, The Netherlands) followed by the SV Total RNA Isolation System (Promega RNA, WI, USA) to prevent any DNA contamination. RT-PCR

Immediately after RNA isolation, 4.5 µg of RNA was used for cDNA synthesis according

to manufacturer’s instructions (Roche Diagnostics). Samples were incubated at 25 °C for

10 minutes, 45 °C for 60 minutes and 95 °C for 5 minutes. PCR results were normalized

to β-actin levels. PCR was done in 50 µl cDNA preparations using primers for the genes

summarized in table 1. mRNA levels of Srebp-1c was measured using Real-Time PCR on a 7700 Sequence Detector (Perkin Elmer, Norwalk, CT) using TaqMan chemicals.

Chapter 3: Dietary carbohydrates and VLDL secretion

68

Table 1: PCR primers. Primers used for semi-quantitative PCR and Real-time PCR (srebp-1c). Product Primers

(5’→ 3’) Product size (bp)

Genbank Acc. number

Ref.

β-Actin sense anti-sense

AAC ACC CCA GCC ATG TAC G ATG TCA CGC ACG ATT TCC C

254 M12481

Apob sense anti-sense

GAC ATG GTG AAT GGA ATC ATG TGA AGA CTC CAG ATG AGG AC

319 M14952 (42)

Apoe sense anti-sense

TGG GAG CAG GCC CTG AAC GCT TC GAG TCG GGC CTG TGC CGC CTG CAC

238 M12414

Mttp sense anti-sense

ATC TGA TGT GGA CGT TGT GT CCT CTA TCT TGT AGG TAG TG

491 L47970 (43)

Dgat sense anti-sense

TGT TCA GCT CAG ACA GTG GT AGG TTG TCT GGA TAG CTC AC

522 AF078752 (43)

Fas sense anti-sense

ATG CCA TGC TGG AGA ACC AG TCT CGG ATG CCT AGG ATG TG

490 X13415

Acc sense anti-sense

GGG ACT TCA TGA ATT TGC TGA TTC TCA GTT GTC ATT ACC ATC TTC ATT ACC TCA ATC TC

729 J03808 (44)

Srebp-1c sense anti-sense TaqMan

GGA GCC ATG GAT TGC ACA TT CCT GTC TCA CCC CCA GCA TA CAG CTC ATC AAC AAC CAA GAC CAGT GAC TTC C

(45)

Abbreviations used: Apo, apolipoprotein; Mttp, microsomal triglyceride transfer protein; Dgat, diacylglycerol acyltransferase; Fas, fatty acid synthase; Acc, acyl Coenzyme A carboxylase; Srebp, sterol regulatory element-binding protein.

HEPATIC MTP PROTEIN LEVELS

Microsomes were isolated from the liver homogenate by centrifugation for 30 min at

10,000 g to remove plasma membranes, for 30 min at 18 000 rpm in a Kontron TST41.14 rotor to remove nuclei and, finally, for 60 min at 28 000 rpm in the same rotor to pellet the microsomes. The pellet thus obtained was resuspended for >17 h in a hypotonic buffer (1 mM Tris (pH 7.4), 0.02% saponin (Sigma, St. Louis, MO)) to release

microsomal lumenal content. Samples were subjected to polyacrylamide gel electrophoresis followed by transfer to a nitrocellulose membrane. MTP and PDI were detected as described before (26) The antibody was kindly provided by dr. C. Shoulders (London, UK). Band intensities were measured using an image densitometer

(Imagemaster, Amersham Pharmacia Biotech, Uppsala, Sweden). MISCELLANEOUS

Protein concentrations were determined according to Lowry (27) using bovine serum

albumin as standard. STATISTICAL ANALYSIS

Comparisons between sucrose and chow diet and between both groups were analyzed

using the non-parametric Mann Whitney U test for independent samples.

The role of apoE in the assembly and secretion of VLDL

69

Figure 1: Liver mRNA levels of lipogenic genes. RNA was isolated and used for PCR as described in the Materials and Methods section. 5 µl of PCR cDNA was subjected to a 2% agarose gel for electroforesis. Band intensities were measured using an image densitometer (Imagemaster, Amersham Pharmacia Biotech). Black bars represent genes from wildtype mice, white bars represent Apoe-/- genes. Expression of the Srebp-1c gene was measured by TaqMan Real-Time PCR. The primers that were used are summarized in table 1.

RESULTS

EFFECTS OF SUCROSE FEEDING ON PLASMA LIPIDS AND INSULIN LEVELS

Feeding mice a 40% sucrose/fat-free diet for two weeks resulted in only marginal

changes in plasma TG and cholesterol levels compared to chow diet (table 2). Plasma TG concentrations were hardly affected, and a small but significant decrease in plasma cholesterol was observed in apoE-deficient mice. Free fatty acids, on the other hand, were strongly decreased in sucrose-fed mice. Remarkably, fasting insulin levels in apoE-

deficient mice were lower than in controls on both diets. HEPATIC LIPID CONTENT AND EXPRESSION OF LIPOGENIC GENES

High-carbohydrate diets have been reported to induce steatosis in rodents under certain

conditions (1,3). As shown in table 3, apoE-deficient mice develop hepatic fat deposits when fed a normal chow diet, as described before (16,17). Sucrose feeding did not lead to a significantly increased hepatic fat content, neither in wildtype nor in apoE-deficient mice. Free cholesterol content was higher in apoE-deficient mice than in wildtype mice

on both diets, as also described before (16,17). In contrast to expectations, mRNA levels of Fas and Acc, i.e., genes involved in

fatty acid synthesis, were not increased after feeding the mice with the sucrose diet.

Expression of these genes even tended to be decreased in apoE-deficient mice fed sucrose and were similar in wildtype animals fed either chow or sucrose (figure 1). Expression of Srebp-1c, the transcription factor critically involved in control of hepatic

lipogenesis, tended to be higher in livers of apoE-deficient mice than those in corresponding wildtypes, but sucrose did not have a significant effect on the expression levels of Srebp-1c in both strains.

0.00

0.50

1.00

1.50

2.00

2.50

3.00

Fas Acc Srebp1c

Rel

ativ

e ex

pres

ion

(com

p. to

β-ac

tin)

C S C S C S

Chapter 3: Dietary carbohydrates and VLDL secretion

70

Table 2: Plasma lipid and insulin concentrations. TG

(mM) cholesterol

(mM) free fatty acids

(mM) insulin (nM)

wildtype chow 0.5 ± 0.1 2.1 ± 0.2 1.75 ± 0.25 0.31 ± 0.11

sucrose 0.6 ± 0.1 2.0 ± 0.1 0.80 ± 0.04 * 0.48 ± 0.17 Apoe-/-

chow 0.8 ± 0.3 16.2 ± 2.3 † 2.09 ± 0.25 0.16 ± 0.02 † sucrose 1.1 ± 0.2 * 12.2 ± 2.0 *† 1.02 ± 0.13 *† 0.12 ± 0.01 *†

Blood from wildtype C57BL/6J and Apoe-/- mice was obtained by heart puncture after a 9 h fast. Plasma TG, cholesterol, free fatty acids, and insulin concentrations were determined by commercially available kits. Mean values ± SD are shown, n=5 in all groups. *) Significant difference (P<0.05) compared to chow diet, †) Significant difference (P<0.05) compared to wildtype on the same diet. Table 3: Hepatic lipid levels. TG

(nmol/mg liver) Free cholesterol (nmol/mg liver)

Cholesteryl esters (nmol/mg liver)

wildtype chow 20.6 ± 6.21 4.2 ± 0.2 1.1 ± 0.1

sucrose 26.1 ± 9.8 3.4 ± 0.1 0.8 ± 0.2 Apoe-/-

chow 64.1 ± 22.8 * 5.2 ± 0.6 * 2.0 ± 1.0 sucrose 66.8 ± 22.9 * 8.8 ± 4.0 * 1.3 ± 1.7

Hepatic lipids from chow-fed and sucrose-fed wildtype C57BL/6J and Apoe-/- mice were analyzed after lipid extraction as detailed under “Materials and Methods”. Values represent the means ± S.D., n = 5 in all groups. *) Significant difference from wildtype mice on the same diet. 1Chow data derived from ref. 17. VLDL-TG SECRETION

VLDL-TG secretion was strongly increased in animals fed the sucrose diet compared to the chow diet (figure 2). This effect was independent from absence of apoE. VLDL-TG

secretion in wildtype mice increased from 165 ± 21 µmol/kg/h on the chow diet to 346

± 29 µmol/kg/h (P<0.05) on the sucrose diet (+109%), while apoE-deficient mice showed

an increase from 93 ± 12 to 217 ± 42 µmol/kg/h (+134%). Thus, VLDL secretion was

impaired in apoE-deficient mice compared to wildtype mice on both diets.

The role of apoE in the assembly and secretion of VLDL

71

0

5

10

15

20

25

30

35

40

0 1 2 3

time after Triton WR 1339 (h)

wtApoe-/-

chowsucrose

Plas

ma

TG (m

M)

*†

†*

Figure 2: VLDL-TG production. Mice were injected intravenously with Triton WR 1339. Plasma TG concentrations during time were determined at regular time intervals. n=5 for each group. *) indicates significant difference in TG production rate between sucrose and chow diet, †) indicates significant difference in TG production rate between wildtype and apoE-deficient mice as calculated from the slope of the curve (P<0.05).

0.00

0.50

1.00

1.50

2.00

Mttp Apoe Apob Dgat

Rel

ativ

e ex

pres

ion

(com

p. to

β -ac

tin)

C S C S C S C S

Figure 3: Liver mRNA levels of genes involved in VLDL secretion. The protocol was essentially the same as in figure 2. Black bars represent genes from wildtype mice, white bars represent Apoe-/- genes.

Chapter 3: Dietary carbohydrates and VLDL secretion

72

HEPATIC EXPRESSION OF GENES INVOLVED IN VLDL ASSEMBLY

To gain insight in the regulation of the apoE-dependent and independent VLDL secretion, mRNA levels of genes involved in hepatic TG metabolism and VLDL assembly were determined by RT-PCR. Some genes involved in VLDL assembly (Apob, Mttp, and

Dgat) tended to be increased in mice fed the sucrose diet, but the differences did not reach statistical significance (figure 3). Apoe mRNA levels were not affected in wildtype

mice fed the sucrose diet. Table 4: VLDL particle size distribution. Before Triton

(nm) After Triton

(nm) mean particle size distribution range mean particle size distribution range wildtype

chow 70.4 19.2 ND1 sucrose 71.8 24.5 79.6 34.6

Apoe-/- chow 55.7 21.2 ND

sucrose 57.2 24.7 85.3 50.0 Particle size of VLDL was determined before and at 4 h after injection of Triton WR 1339. Size and size distribution were analyzed by dynamic light scattering. Diameters were obtained by volume distribution provided by the analyzer. Distribution was determined for n = 2 per diet and group. 1ND, not determined. VLDL SIZE AND COMPOSITION

By subtracting steady state VLDL composition from VLDL composition 4 h after Triton

WR 1339 injection, nascent VLDL composition was estimated (23). As shown in figure 4, VLDL composition was hardly affected by diet or genotype. There was a tendency for an increased TG content per particle after sucrose feeding, but this effect was not statistically significant. Steady state VLDL size was decreased in apoE-deficient mice

presumably due to the longer circulation time in plasma. This difference disappeared after injection of Triton WR-1339, which prevents hydrolysis of the lipoproteins. As shown in table 4, VLDL size as determined by dynamic light scattering was not changed by the sucrose diet.

The role of apoE in the assembly and secretion of VLDL

73

TGFC

������ CE���

PL��������������

76 ± 18 %

13 ± 10 %

11 ± 7 %

��������������

72 ± 18 %15 ± 6 %

12 ± 4 %A �������������� 71 ± 13 %

19 ± 20 %

10 ± 7 %B

���������������������������������� 76 ± 13 %

10 ± 2 %

2 ± 1 %

12 ± 2 %C D

Figure 4: VLDL composition. VLDL from fasted wildtype and apoE-deficient mice before and four hours after intravenous injection of triton WR-1339 was isolated from plasma (d<1.019 g/ml) by ultracentrifugation. TG and phospholipids were determined using commercially available kits (Roche Diagnostics and WAKO Chemicals). The difference between the composition 4 h after and before triton injection was used to estimate nascent VLDL composition. TG, triglycerides; FC, free cholesterol; CE, cholesteryl ester; PL, phospholipids. A) wildtype chow, B) Apoe-/- chow, C) wildtype sucrose, D) Apoe-/- sucrose.

A

wildtype Apoe-/- wildtype Apoe-/-

chow sucrose

MTP

PDI

B

0.0

0.0

0.2

0.3

0.4

0.5

0.6

wt Apoe-/-

* *

Rel

ativ

e M

TP/ P

DI l

evel

s

Figure 5: MTP protein levels. Hepatic microsomes from chow- and sucrose-fed wildtype and apoE-deficient mice were isolated by ultracentrifugation. Lumenal content was released using a hypotonic buffer in the presence of 0.02% saponin. Samples were applied to gel and blotted to nitrocellulose by Western blot. MTP and PDI were detected using an antibody against human MTP/PDI cross-reactive with murine MTP/PDI. A) Western blot of 3 representative samples per group. B) Intensities of MTP were related to the abundant PDI protein. Band intensities were measured using an image densitometer (Imagemaster, Amersham Pharmacia Biotech), n=3 in all groups. *) Significant difference (P=0.05) compared to chow diet.

Chapter 3: Dietary carbohydrates and VLDL secretion

74

MTP PROTEIN LEVELS

Figure 5 shows that protein levels of MTP were clearly increased in liver microsomes of mice fed the sucrose diet compared to the chow diet. This effect was apoE-genotype-independent, as MTP levels were comparable in both strains of mice under both dietary

conditions. The decreased VLDL-TG secretion in apoE-deficient mice compared to wildtype mice is therefore not due to decreased MTP levels. EFFECTS OF DIETARY FRUCTOSE ON PLASMA AND HEPATIC LIPID METABOLISM IN MICE

To verify whether the effect of dietary sucrose on lipid metabolism was specifically due to the sucrose – consisting of a glucose and a fructose unit – some lipid parameters were also determined in mice fed a liquid fructose diet in addition to their regular chow diet. As shown in figure 6a, dietary fructose caused a strong increase in plasma TG

levels, an effect that has been described before (20). This effect was seen in both strains. Comparable to the situation observed after feeding the sucrose diet, hepatic lipid levels did not change in mice fed fructose (figure 6b). In addition, fructose led to a modest effect on VLDL secretion (figure 6c). Gene expression of Fas was increased in apoE-

deficient mice only (from 90 ± 20 % to 310 ± 180 % compared to wildtype mice on

chow).

DISCUSSION

Reduction of dietary fat intake, as propagated in health promotion campaigns, is typically associated with an increased dietary carbohydrate content. The shift from fat to

carbohydrates as main energy source is usually associated with a decrease in plasma cholesterol levels, but frequently also accompanied by increased plasma TG levels. Elevated plasma TG has been recognised as an independent risk factor for atherosclerosis (8). Humans carrying the E2 allele of the APOE gene have a greater

increase in plasma TG levels during high dietary sucrose intake than those with the E3 or E4 allele (28). We (15-17) and others (29-31) have shown that apoE is involved in the control of VLDL-TG secretion. Furthermore, there are data suggesting that Apoe

expression in rodent liver is increased by high carbohydrate intake (18). Therefore we tested the hypothesis that high carbohydrate intake modulates hepatic VLDL production via an apoE-mediated metabolic route.

In our study, mice fed the high-sucrose diet for two weeks did not develop a fatty

liver, in contrast to other reports (1,4). However, absence of hepatic fat accumulation in mice under similar conditions has also been reported (6,7,32). The use of different diets and/or mouse strains may contribute to the divergent results. The expression of the

The role of apoE in the assembly and secretion of VLDL

75

% o

f wild

-type

on

chow

die

t

02040

6080

100120

140

160

VLDL-TG PR

wt Apoe-/-

*

*

C

0

100

200

300

400

500

TG FC CE

B

wt Apoe-/-wt Apoe-/-wt Apoe-/-

0

200

400

600

800

1000

1200

TG Cholesterol

* *

A

wt Apoe-/- wt Apoe-/-

% o

f wild

-type

on

chow

die

t

% o

f wild

-type

on

chow

die

t

Figure 6: Effect of dietary fructose on lipid metabolism. A) Plasma lipid levels, B) Hepatic lipid levels, C) VLDL-TG production rate. For comparison, all levels are expressed as % of wildtype mice on chow diet. Black bars corresponds to the chow diet, white bars corresponds to the fructose diet. *) Significant difference (P=0.05) compared to chow diet.

Chapter 3: Dietary carbohydrates and VLDL secretion

76

genes encoding enzymes involved in lipogenesis, such as Fas and Acc, were not increased after feeding mice the sucrose-containing diet: in apoE-deficient mice Fas and Acc mRNA levels even tended to be decreased. The expression of the transcription

factor Srebp-1c, a key factor in regulation of genes involved in lipogenesis (7), was also not affected. Absence of effects on “lipogenic” gene expression probably explain the absence of carbohydrate-induced hepatic steatosis in our study. It may be that the genes

were initially upregulated as it has been reported that Fas enzyme activity is highest during the first 3-4 days after the start of the diet, followed by a slow decrease of activity (33). Development of a fatty liver in rats has been associated with age. Only adult animals developed a fatty liver upon carbohydrate feeding which was associated with

the inability to induce VLDL-TG secretion. Young rats, on the other hand, did show increased VLDL-TG secretion and did not develop steatosis. In our set-up, VLDL-TG secretion was strongly increased by dietary carbohydrates in C57BL/6J wildtype mice as well as in apoE-deficient mice, i.e., independent of apoE-genotype. Upregulation of Apoe

gene expression has been reported after fasting-refeeding carbohydrates in rats in vivo (18) and in vitro in sucrose-fed rat hepatocytes (34,35). In our studies, in which the mice

were fed a sucrose diet for 2 weeks, no increase in Apoe mRNA could be detected. This supports the conclusion that carbohydrate-induced VLDL-TG secretion is independent from apoE. However, VLDL-TG secretion remained reduced in carbohydrate-fed apoE-

deficient mice compared to carbohydrate-fed wildtype mice. This finding implies that apoE and dietary carbohydrates affect VLDL-TG secretion by different mechanisms. Expression of genes encoding proteins that are critically involved in VLDL-TG secretion, i.e., Dgat, Mttp and Apob, tended to be increased by dietary sucrose, and MTP-protein

levels were markedly increased. The latter is in agreement with a report of Taghibiglou et

al. (36) showing that increased VLDL-TG secretion in fructose-fed Syrian golden hamsters

was associated with increased MTP protein levels. Lin et al. (37) have shown that a high sucrose diet results in a 55% increase in MTP gene expression in this species.

Although VLDL-TG secretion in apoE-deficient mice was strongly decreased compared to wildtype mice, MTP protein levels were similarly affected by carbohydrate feeding. This finding indicates that apoE mediates its effect on VLDL secretion relatively late in the secretory pathway, i.e., after the actions mediated by MTP. In this scenario, an

increase in MTP protein levels may lead to an overall induction of VLDL secretion, which remains lower in apoE-deficient mice compared to wildtype mice because the “late apoE effects” are absent. As we only observed increased protein levels of MTP without a substantial increase in Mttp gene expression, our data indicate a post-translational

regulation of MTP protein levels. MTP is a very stable protein, with a half-life of 4.4 days. Changes in gene expression will therefore not acutely affect protein levels (38).

The role of apoE in the assembly and secretion of VLDL

77

The results from the high sucrose diet were confirmed in an experiment in which the drinking water of wildtype C57BL/6J and apoE-deficient mice was supplemented with 10% fructose. This feeding regime lead to a modest increase in VLDL-TG secretion, and to a strong increase in plasma TG. The latter effect appears to be a specific fructose-

mediated metabolic effect caused by decreased lipoprotein hydrolysis and hepatic lipoprotein uptake (5) and increased de novo lipogenesis mediated by specific activation of pyruvate dehydrogenase (PDH) (20,39). In addition, fructose by-passes the phosphofructokinase regulatory step in glycolysis, which may contribute to increased

lipogenesis (40). Fas expression in fructose-fed C57BL/6J and apoE-deficient mice was modestly increased, but again this did not result in hepatic lipid accumulation.

The exact mechanism of dietary carbohydrate-induced VLDL secretion is not

known. Carbohydrates can be metabolized in the glycolytic pathway to form glycerol-3-phosphate, the substrate for the phosphatidate route of TG synthesis. The fatty acids required for esterification of glycerol-3-phosphate to form TG may be derived from de

novo lipogenesis but also from adipose tissue (4). The fatty acid flux from adipocytes to the liver is controlled, to a large extent, by prevailing plasma insulin concentrations. Low plasma insulin levels and resistance of adipocytes to the metabolic actions of insulin may

lead to an increased fatty acid flux to the liver. In combination with a decreased β-

oxidation due to malonyl CoA-induced inhibition of CPT-1 activity (41), this may lead to a relative excess of hepatic TG (4). Plasma free fatty acid levels in wildtype and apoE-deficient mice were decreased in sucrose-fed mice. This, however, does not exclude the

possibility that fatty acid flux was increased, perhaps during certain periods of the day. Based on our results, we propose that VLDL-TG secretion is stimulated in

C57BL/6J mice after ingestion of a low-fat/high-carbohydrate diet for two weeks, independent from effects on lipogenesis and without development of a fatty liver.

Induction of MTP protein levels may be of crucial importance in this respect. In addition, carbohydrate-stimulated VLDL-TG secretion does not require the presence of apoE. Our results indicate that the cascade of events in VLDL-TG secretion involves specific apoE-independent and apoE-dependent components.

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