Gene Expression in Livers of BALB/C and C57BL/6J Mice Fed a High-Fat Diet

Abstract

We previously demonstrated that high-fat diet (HFD)–induced hepatic lipid accumulation is more severe in BALB/c mice than in C57BL/6J (B6) mice. To understand the changes in liver metabolism, we studied blood chemistry, gene expression, and histopathological changes of the liver in nine-week HFD-fed BALB/c and B6 mice and one- or four-week HFD-fed BALB/c mice. Serum total cholesterol and triglyceride levels were significantly increased in all HFD-fed groups, and one- and four-week HFD-fed BALB/c groups, respectively. Histopathology revealed that vacuolation of hepatocytes was severe in nine-week HFD-fed BALB/c mice, although it was less severe in the other groups. Microarray analysis of mRNA expression of nine-week HFD-fed BALB/c mice showed up-regulation of genes involved in fatty acid uptake and biosynthesis, such as Cd36, Acaca, Acly, and Fasn. Some changes were observed in the one- and four-week HFD-fed BALB/c groups and the nine-week HFD-fed B6 group, however these changes in mRNA expression were not so marked. In conclusion, the fatty accumulation observed in BALB/c mice may be caused, at least in part, by up-regulation of fatty acid uptake and biosynthesis. Cd36, Acaca, Acly and Fasn may be involved in these metabolic processes.

Keywords

high-fat diet liver microarray steatosis mouse

Introduction

Hepatic lipid metabolism is influenced by the balance between the degradation and synthesis and/or import and export of triacylglycerol (TAG) and fatty acids. Disruption of this balance causes hepatic steatosis.

In laboratory animals, many models of hepatic steatosis have been created. Among genetically modified animal models, acyl-coenzyme A oxidase null mice, methionine adenosyltransferase-1A null mice, nuclear respiratory factor 1 knockout mice, phosphatase and tensin homolog deleted on chromosome 10 null mice, ob/ob mice, and db/db mice have been reported (Fan and Qiao 2009). Among dietary mouse models, there are methionine- and choline-deficient diet–induced and high-fat diet–induced models (Fan and Qiao 2009). Moreover, combined mouse models induced by the combination of genetic modification and nutritional/dietary challenges have also been reported (Fan and Qiao 2009). In zebrafish, exposure of larvae to alcohol leads to hepatic steatosis in a similar manner to that observed in humans (Passeri et al. 2009). In neonatal piglets, total parenteral nutrition induces liver steatosis and apoptosis (Wang et al. 2006). There are also ethanol- and protein-deficient diet–induced steatotic liver models in pigs (Spannbaure et al. 2005). In particular, in view of their usability and the similarity to human diet–induced steatosis, high-fat diet (HFD)–induced rodent models are useful tools for characterizing the pathophysiological and molecular mechanisms involved in the progression of hepatic steatosis.

West et al. reported that C57BL/6J (B6) mice are a particularly obesity-prone strain among nine different inbred mouse strains examined (West et al. 1992). B6 mice have been used in many studies as obesity and steatosis models, whereas A/J mice have been used as an obesity-resistant mouse strain in some reports.

In fatty liver disease, various potential candidate genes have been reported, including sterol regulatory element binding factor (Srebf) 1c, stearoyl CoA desaturase-1 (Scd1), microsomal triglyceride transfer protein (Mttp), phosphatidylethanolamine methyltransferase, and peroxisome proliferator-activated receptor (PPAR)-α (Wilfred de Alwis and Day 2007). Several studies analyzing gene expression in animal models of HFD-induced hepatic steatosis have been reported. However, many of these reports examined only one time-point or did not include any histopathology.

We previously observed that HFD-induced hepatic lipid accumulation is more severe in BALB/c mice than in B6 mice (Nishikawa et al. 2007; Nishikawa et al. 2008). In this study we analyzed changes in gene expression and histopathological patterns as well as important metabolic changes underlying the development of hepatic steatosis.

Materials and Methods

One- and Four-Week-Fed BALB/c Groups

Animals

Nine-week-old and twelve-week-old male BALB/cAJcl mice were used for these experiments. The animals, purchased from CLEA Japan, were housed individually in plastic mouse cages and maintained under controlled conditions (twelve-hour light:dark cycle, temperature of 23°C ± 3°C, relative humidity of 50% ± 20%). For acclimatization, mice were fed a standard rodent chow (CE-2, CLEA Japan, Tokyo, Japan), with ad libitum access to water for one week. After this period, the ten-week-old mice were fed either High Fat Diet 32 (HFD32, CLEA Japan), or CE-2 for four weeks. The thirteen-week-old mice were fed in the same way for one week. The composition of each of the two diets is shown in Table 1 . At the end of the experiment, all the mice were fourteen weeks old. They were fasted for twenty hours and were then exsanguinated from the caudal vena cava under ether anesthesia, and the liver and peri-epididymal fat tissues were collected and weighed. The left lateral lobe of the liver was fixed in 10% neutral buffered formalin for histopathology, whereas the right and caudate lobes were stored at –80°C for RNA extraction. All these procedures were performed according to the “Rules for Feeding and Storage of Experimental Animals and Animal Experiments” and were approved, from the point of view of animal welfare, by the Institutional Animal Care and Use Committee of the testing facility.

Table 1.

Diet composition.

	HFD32	CE-2
Moisture (%)	6.9	9.2
Protein (%)	25	25.6
Fat (%)	32.4	4
Fiber (%)	2.9	3.8
Ash (%)	4	6.9
Nitrogen-free extract (%)	28.8	50.5
Energy (kcal/100 g)	506.8	340.4
Fat-derived energy (%)	57.5	10.6
Protein-derived energy (%)	19.7	30.1

Blood Biochemistry

Serum samples were prepared from the blood collected through the caudal vena cava by centrifugation at 2,000–3,000 rpm for ten minutes. Serum TAG, total cholesterol (Cho), glucose, and phospholipid levels were determined using a Hitachi 7180 autoanalyzer (Hitachi High-Technologies Corp., Tokyo, Japan).

Nine-Week–Fed BALB/c and B6 Groups

The method used has been previously described in detail (Nishikawa et al. 2008). Briefly, five-week-old male BALB/cAJcl and C57BL/6JJcl mice were fed a CE-2 or HFD32 diet for nine weeks. At the end of the experiment, mice were fasted for twenty hours, then blood, liver, and peri-epididymal fat tissues were collected. The left lateral lobe of the liver was fixed in 10% neutral buffered formalin for histopathology, whereas the right and caudate lobes were stored at –80°C for RNA extraction. Serum Cho, TAG, and plasma glucose were measured using commercially available kits (Cholesterol E-test Wako, Triglyceride E-test Wako, and Glucose CII-test Wako; all from Wako, Osaka, Japan). All procedures involving mice were conducted according to the guidelines for the care and use of laboratory animals approved by the Graduate School of Agricultural and Life Sciences of Tokyo University. The following methods were used in the one- or four-week–fed BALB/c groups and the nine-week–fed BALB/c and B6 groups.

Histopathological Examination

The liver samples fixed in 10% neutral buffered formalin were dehydrated and embedded in paraffin. Four-µm-thick paraffin sections were stained with hematoxylin and eosin.

Calculation of Energy Intake

Dietary intake was obtained by subtracting the weight of the remaining feed from the weight of the feed initially supplied. Energy intake was calculated on the basis of energy values of 3.404 kcal/g and 5.068 kcal/g for the CE-2 and the HFD feed, respectively.

Statistical Analysis

Results are presented as mean ± standard deviation (SD). The Student t test was used to compare HFD-fed groups with control groups.

Isolation of Total RNA

Each liver sample of approximately 30 mg was lysed in 600 µL of RLT buffer (QIAGEN Co. Ltd., Tokyo, Japan) using a Mixer Mill (QIAGEN). Total RNA was extracted from the liver homogenate using RNeasy Plus (QIAGEN) according to the manufacturer’s protocol. The ratio of absorbance at 260 nm and 280 nm (A260/A280) was calculated for each RNA sample from measurements taken using a NanoDrop ND-1000 spectrophotometer (Isogen, Maarssen, The Netherlands). The integrity of total RNA was checked by the RNA integrity number generated by a 2100 Bioanalyzer (Agilent Technologies, Amsterdam, The Netherlands) with 6000 Nano Chips according to the manufacturer’s instructions. Since the A260/A280 and RNA integrity numbers were all above 1.8 and 7, respectively, this finding confirmed that all the total RNA samples were appropriate for use in the subsequent GeneChip experiment. Two RNA samples from each group were used for hybridization to microarrays.

Preparation of Biotin-Labeled cRNA for Hybridization

Two µg of total RNA was used for the preparation of biotin-labeled cRNA for hybridization. The cRNA was prepared according to the manufacturer’s instructions using the GeneChip Array Station (Affymetrix).

Hybridization and Signal Detection

A GeneChip Mouse Genome 430 2.0 Array (Affymetrix) cartridge was filled with the hybridization cocktail, and then incubated at 45°C overnight. Arrays were then washed and stained in a GeneChip Fluidics Station 450 (Affymetrix) according to Fluidics Protocol FS450_0001, and the hybridization signal was detected by a GeneChip Scanner 3000 (Affymetrix). The scanned images were processed by GeneChip operating software (Affymetrix).

Data Normalization

GeneChip data were normalized using GeneSpring Gx 7.3.1 (Agilent Technologies). After data transformation (set measurements < 0.01–0.1), per-chip normalization was performed to equalize the 50th percentile of each GeneChip (normalize to 50th percentile). After this step, the intensities were normalized for each gene by setting the mean intensity to mean signal of control chips.

Data Analysis

Extraction of Affected Genes

Genes that exhibited changes greater than 1.5-fold were categorized as up-regulated (in the HFD-fed animals, genes that exhibited the “present call” (labeled as present by Affymetrix detection call) were subjected to fold-change analysis. Genes that exhibited changes less than 0.67-fold were categorized as down-regulated (in the CE-2-fed animals, genes that exhibited the “present call” were subjected to fold-change analysis).

Identification of Lipid Metabolism-Related Genes

Based on the KEGG PATHWAY (Kyoto Encyclopedia of Genes and Genomes), the genes related to fatty acid biosynthesis, fatty acid elongation in mitochondria, biosynthesis of unsaturated fatty acids, steroid biosynthesis, terpenoid backbone biosynthesis, glycerolipid metabolism, synthesis and degradation of ketone bodies, primary bile acid biosynthesis, and the PPAR-α signaling pathway were selected from those genes that were differentially expressed. From the genes involved in PPAR-α signaling, those related to adipocyte differentiation, adaptive thermogenesis, cell survival, ubiquitination, and gluconeogenesis were excluded. In addition, important genes involved in fatty acid biosynthesis, ATP-citrate lyase (Acly); lipid influx, low-density lipoprotein receptor (Ldlr), very low-density lipoprotein receptor (Vldlr), and scavenger receptor class B member 1 (Scarb1); lipid efflux, apolipoprotein B (ApoB), Mttp, ATP-binding cassette subfamily A member 1 (Abca1), as well as the transcription factors Srebf1 and Srebf2, were also analyzed.

Results

Results of the nine-week–fed BALB/c and B6 groups and the one- and four-week–fed BALB/c groups are presented together.

Energy Intake, Body Weight, and Organ Weight

The energy intake, body weight, and organ weight of the animals in the study are shown in Table 2 . The total energy intake and body weight of HFD-fed mice were significantly greater than those of control mice in the one-week–fed BALB/c group and in the nine-week–fed BALB/c and B6 groups. In all HFD-fed groups, the energy intake was higher than that of the respective control groups during the first few days of the feeding period followed by a decrease to control levels (data not shown); therefore, the increased energy intake and body weight in the one-week–fed BALB/c group may be transient. The liver weight was increased only in the nine-week–fed BALB/c group, but it was not significant with respect to relative liver weight. The reason for the decrease in relative liver weight in the four-week–fed BALB/c group was not clear. In the nine-week–fed B6 group, the relative liver weight decreased because the liver weight was unchanged and body weight had markedly increased. The peri-epididymal fat weight had increased in all HFD-fed groups. Between the nine-week–fed BALB/c and B6 groups, the fat weight was almost equal.

Table 2.

Energy intake, body weight, and organ weight.

		Control	HFD
Total energy intake (kcal)	One-week–fed BALB	92.15 ± 4.51	104.04 ± 3.28 **
	Four-week–fed BALB	426.60 ± 8.56	434.67 ± 77.55
	Nine-week–fed BALB	836.53 ± 34.27	1006.50 ± 89.00 **
	Nine-week–fed B6	636.72 ± 33.91	738.41 ± 37.23 **
Body weight (g)	One-week–fed BALB	24.20 ± 0.65	26.22 ± 0.69 **
	Four-week–fed BALB	27.24 ± 0.80	29.42 ± 2.14
	Nine-week–fed BALB	26.88 ± 1.04	36.10 ± 2.39 **
	Nine-week–fed B6	22.66 ± 0.99	32.02 ± 3.64 **
Liver weight (g)	One-week–fed BALB	1.13 ± 0.08	1.22 ± 0.05
	Four-week–fed BALB	1.29 ± 0.09	1.16 ± 0.12
	Nine-week–fed BALB	1.27 ± 0.05	1.83 ± 0.24 **
	Nine-week–fed B6	1.02 ± 0.03	1.14 ± 0.18
Liver weight / body weight (%)	One-week–fed BALB	4.65 ± 0.25	4.64 ± 0.11
	Four-week–fed BALB	4.74 ± 0.40	3.98 ± 0.17 **
	Nine-week–fed BALB	4.74 ± 0.07	5.06 ± 0.35
	Nine-week–fed B6	4.50 ± 0.20	3.56 ± 0.23 **
Periepididymal fat weight (mg)	One-week–fed BALB	107 ± 43	395 ± 50 **
	Four-week–fed BALB	107 ± 34	589 ± 192**
	Nine-week–fed BALB	237.0 ± 58	14389 ± 356 **
	Nine-week–fed B6	217 ± 31	1504 ± 360 **
Periepididymal fat weight / body weight (%)	One-week–fed BALB	0.44 ± 0.17	1.51 ± 0.21 **
	Four-week–fed BALB	0.39 ± 0.13	1.97 ± 0.56 **
	Nine-week–fed BALB	0.88 ± 0.19	3.95 ± 0.74 **
	Nine-week–fed B6	0.96 ± 0.13	4.66 ± 0.75 **

Values are mean ± SD; n = 4–6.

Significant differences are indicated by * and ** at p <.05 and p < .01, respectively.

Blood Biochemistry

As shown in Table 3 , total Cho levels were significantly increased in all HFD-fed groups. The TAG and phospholipid levels in the one- and four-week–fed BALB/c groups and the glucose level in the nine-week–fed BALB/c group were also all significantly higher. For phospholipids, only the one- and four-week–fed BALB/c groups were examined.

Table 3.

Blood chemistry.

		Control	HFD
Total cholesterol (mg/dL)	One-week–fed BALB	76.8 ± 7.0	111.6 ± 9.3 **
	Four-week–fed BALB	82.4 ± 13.6	145.8 ± 20.4 **
	Nine-week–fed BALB	79.3 ± 7.3	129.0 ± 26.8 *
	Nine-week–fed B6	69.3 ± 7.0	101.7 ± 7.8 **
Triglyceride (mg/dl)	One-week–fed BALB	20.4 ± 14.5	55.0 ± 10.6 **
	Four-week–fed BALB	16.6 ± 5.6	54.8 ± 27.2 *
	Nine-week–fed BALB	70.3 ± 21.1	120.8 ± 65.3
	Nine-week–fed B6	46.7 ± 15.6	43.7 ± 11.1
Glucose (mg/dl)	One-week–fed BALB	130.2 ± 14.8	142.6 ± 43.1
	Four-week–fed BALB	152.0 ± 13.5	171.0 ± 29.6
	Nine-week–fed BALB	126.6 ± 25.9	144.1 ± 27.9
	Nine-week–fed B6	155.9 ± 20.3	215.4 ± 36.0 **
Phospholipid (mg/dl)	One-week–fed BALB	150.4 ± 16.6	218.6 ± 15.6 **
	Four-week–fed BALB	157.8 ± 31.0	255.0 ± 36.4 **
	Nine-week–fed BALB	–	–
	Nine-week–fed B6	–	–

-: not examined. Values are means ± SD; n = 4–6.

Significant differences are indicated by * and ** at p < .05 and p < .01, respectively.

Histopathology

In all control (CE-2–fed) groups, no vacuolation of hepatocytes was observed. In the HFD groups, the severity of vacuolation of hepatocytes was moderate in the nine-week–fed BALB/c group, and mild in the one- and four-week–fed BALB/c groups and the nine-week–fed B6 group. In the four-week–fed BALB/c mice and the nine-week–fed BALB/c and B6 mice, macrovacuoles were observed (inset of Figure 1 ).

Figure 1.

Photomicrographs of hematoxylin and eosin–stained liver sections. Scale bar = 200 μm (20 μm for insert). (A) One-week CE-2–fed BALB/c mice. (B) Nine-week CE-2–fed B6 mice. (C) One-week HFD-fed BALB/c mice. (D) Nine-week HFD-fed B6 mice. (E) Four-week HFD-fed BALB/c mice. (F) Nine-week HFD-fed BALB/c mice. Insets show a higher magnification of the respective figure. Vacuolation of hepatocytes was severe in the nine-week HFD-fed BALB/c mice. In four-week HFD-fed BALB/c mice and nine-week HFD-fed B6 mice, a few macrovacuoles were observed (inset).

Microarray Analysis (Table 4 )

Table 4.

List of genes differentially expressed under the influence of a high-fat diet in one-, four-, and nine-week–fed B6 mice.

Affymetrix ID	Abbreviation	Gene Title	KEGG PATHWAY	Fold
Affymetrix ID	Abbreviation	Gene Title	KEGG PATHWAY	1wBALB	4wBALB	9wBALB	9wB6
Fatty acid biosynthesis
1444810_at	Acaca	acetyl-Coenzyme A carboxylase alpha	Fatty acid biosynthesis	–	–	1.72	1.51
1439445_x_at	Acly	ATP citrate lyase	Not applicable	–	–	1.64	–
1423828_at	Fasn	fatty acid synthase	Fatty acid biosynthesis	–	–	1.70	–
1452216_at	Mcat	similar to mitochondrial malonyltransferase isoform b precursor; malonyl CoA:ACP acyltransferase (mitochondrial)	Fatty acid biosynthesis	–	–	0.65	–
1430307_a_at	LOC677317 /// Me1	similar to NADP-dependent malic enzyme (NADP-ME) (Malic enzyme 1); malic enzyme 1, NADP(+)-dependent, cytosolic	PPAR signaling pathway	0.56	0.52	–	0.60
1416632_at	LOC677317 /// Me1	similar to NADP-dependent malic enzyme (NADP-ME) (Malic enzyme 1); malic enzyme 1, NADP(+)-dependent, cytosolic	PPAR signaling pathway	0.60	–	–	0.51
1442732_at	Hadhb	hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme A thiolase/enoyl-Coenzyme A hydratase (trifunctional protein), beta subunit	Fatty acid elongation in mitochondria	–	–	1.59	–
1422468_at	Ppt1	palmitoyl-protein thioesterase 1	Fatty acid elongation in mitochondria	0.65	–	–	–
1418302_at	Ppt2	palmitoyl-protein thioesterase 2	Fatty acid elongation in mitochondria	–	0.65	–	–
1422996_at	Acot2	acyl-CoA thioesterase 2	Biosynthesis of unsaturated fatty acids	–	–	2.83	–
1439478_at	Acot2	acyl-CoA thioesterase 2	Biosynthesis of unsaturated fatty acids	–	–	1.63	–
1422925_s_at	Acot3	acyl-CoA thioesterase 3	Biosynthesis of unsaturated fatty acids	–	7.74	–	0.38
1443147_at	Acot3	acyl-CoA thioesterase 3	Biosynthesis of unsaturated fatty acids	–	12.10	1.53	0.28
1422076_at	Acot4	acyl-CoA thioesterase 4	Biosynthesis of unsaturated fatty acids	–	1.94	1.65	0.58
1422077_at	Acot4	acyl-CoA thioesterase 4	Biosynthesis of unsaturated fatty acids	–	2.35	–	0.54
1449968_s_at	Acot10 /// Acot9	acyl-CoA thioesterase 9	Biosynthesis of unsaturated fatty acids	0.66	–	–	–
1444518_at	Acox1	acyl-Coenzyme A oxidase 1, palmitoyl	Biosynthesis of unsaturated fatty acidsPPAR signaling pathway	–	–	2.89	–
1415840_at	Elovl5	ELOVL family member 5, elongation of long chain fatty acids (yeast)	Biosynthesis of unsaturated fatty acids	–	–	1.56	–
1440444_at	Fads1	fatty acid desaturase 1	Biosynthesis of unsaturated fatty acids	2.19	–	2.12	–
1419031_at	Fads2	fatty acid desaturase 2	Biosynthesis of unsaturated fatty acidsPPAR signaling pathway	1.54	–	1.63	–
1449325_at	Fads2	fatty acid desaturase 2	Biosynthesis of unsaturated fatty acidsPPAR signaling pathway	–	–	1.55	–
1459530_at	Hsd17b12	hydroxysteroid (17-beta) dehydrogenase 12	Steroid hormone biosynthesisBiosynthesis of unsaturated fatty acids	–	–	4.46	–
1439167_at	Pecr	peroxisomal trans-2-enoyl-CoA reductase	Biosynthesis of unsaturated fatty acids	–	–	2.65	–
1449342_at	Ptplb	protein tyrosine phosphatase-like (proline instead of catalytic arginine), member b	Biosynthesis of unsaturated fatty acids	0.65	–	–	–
1415964_at	Scd1	stearoyl-Coenzyme A desaturase 1	Biosynthesis of unsaturated fatty acidsPPAR signaling pathway	0.23	0.49	–	–
1415965_at	Scd1	stearoyl-Coenzyme A desaturase 1	Biosynthesis of unsaturated fatty acidsPPAR signaling pathway	0.16	0.37	–	–
1415822_at	Scd2	stearoyl-Coenzyme A desaturase 2	Biosynthesis of unsaturated fatty acidsPPAR signaling pathway	–	1.53	2.36	–
1415824_at	Scd2	stearoyl-Coenzyme A desaturase 2	Biosynthesis of unsaturated fatty acidsPPAR signaling pathway	–	–	1.84	–
1436976_a_at	Yod1	YOD1 OTU deubiquitinating enzyme 1 homologue (S. cerevisiae)	Biosynthesis of unsaturated fatty acids	0.53	–	1.77	–
Glycerolipid metabolism
1428821_at	Agpat2	1-acylglycerol-3-phosphate O-acyltransferase 2 (lysophosphatidic acid acyltransferase, beta)	Glycerolipid metabolism	–	–	1.89	–
1422841_at	Agpat6	1-acylglycerol-3-phosphate O-acyltransferase 6 (lysophosphatidic acid acyltransferase, zeta)	Glycerolipid metabolism	–	0.62	–	–
1436276_at	Agpat6	1-acylglycerol-3-phosphate O-acyltransferase 6 (lysophosphatidic acid acyltransferase, zeta)	Glycerolipid metabolism	0.43	0.57	–	–
1437133_x_at	Akr1b3	aldo-keto reductase family 1, member B3 (aldose reductase)	Glycerolipid metabolism	1.60	–	1.81	–
1456590_x_at	Akr1b3	aldo-keto reductase family 1, member B3 (aldose reductase)	Glycerolipid metabolism	–	–	1.56	–
1448894_at	Akr1b8	aldo-keto reductase family 1, member B8	Glycerolipid metabolism	–	1.55	–	–
1417257_at	Cel	carboxyl ester lipase	Steroid biosynthesisGlycerolipid metabolism	–	–	15.57	–
1425300_at	Dak	dihydroxyacetone kinase 2 homolog (yeast)	Glycerolipid metabolism	–	–	–	0.44
1422677_at	Dgat2	diacylglycerol O-acyltransferase 2	Glycerolipid metabolism	–	1.52	–	–
1422678_at	Dgat2	diacylglycerol O-acyltransferase 2	Glycerolipid metabolism	1.67	–	–	–
1418578_at	Dgka	diacylglycerol kinase, alpha	Glycerolipid metabolism	0.64	–	–	–
1438310_at	Dgkh	diacylglycerol kinase, eta	Glycerolipid metabolism	0.39	–	–	1.58
1424995_at	Glyctk	glycerate kinase	Glycerolipid metabolism	–	0.58	–	0.67
1425834_a_at	Gpam	glycerol-3-phosphate acyltransferase, mitochondrial	Glycerolipid metabolism	0.53	–	–	–
1422703_at	Gyk	glycerol kinase	Glycerolipid metabolismPPAR signaling pathway	1.68	–	1.77	–
1422704_at	Gyk	glycerol kinase	Glycerolipid metabolismPPAR signaling pathway	1.57	–	1.53	–
1434690_at	Lclat1	lysocardiolipin acyltransferase 1	Glycerolipid metabolism	–	0.66	–	–
1419560_at	Lipc	lipase, hepatic	Glycerolipid metabolism	–	–	–	1.82
1421262_at	Lipg	lipase, endothelial	Glycerolipid metabolism	–	–	2.99	–
1450188_s_at	Lipg	lipase, endothelial	Glycerolipid metabolism	0.46	2.12	3.39	–
1426785_s_at	Mgll	monoglyceride lipase	Glycerolipid metabolism	–	–	2.70	–
1450391_a_at	Mgll	monoglyceride lipase	Glycerolipid metabolism	–	–	1.61	–
1453836_a_at	Mgll	monoglyceride lipase	Glycerolipid metabolism	–	–	1.72	–
1421868_a_at	Pnlip	pancreatic lipase	Glycerolipid metabolism	–	–	13.91	–
1433431_at	Pnlip	pancreatic lipase	Glycerolipid metabolism	–	–	30.72	–
1415777_at	Pnliprp1	pancreatic lipase related protein 1	Glycerolipid metabolism	–	–	107.87	–
1446850_at	Ppap2b	phosphatidic acid phosphatase type 2B	Glycerolipid metabolism	–	2.29	–	–
1448908_at	Ppap2b	phosphatidic acid phosphatase type 2B	Glycerolipid metabolism	–	–	1.51	–
1451210_at	Ppap2c	phosphatidic acid phosphatase type 2C	Glycerolipid metabolism	–	0.64	0.65	–
Terpenoid and steroid biosynthesis
1418129_at	Dhcr24	24-dehydrocholesterol reductase	Steroid biosynthesis	–	–	2.47	0.66
1438322_x_at	Fdft1	predicted gene 6781; farnesyl diphosphate farnesyl transferase 1	Steroid biosynthesis	–	2.05	–	–
1448130_at	Fdft1	predicted gene 6781; farnesyl diphosphate farnesyl transferase 1	Steroid biosynthesis	–	2.20	–	–
1417871_at	Hsd17b7	hydroxysteroid (17-beta) dehydrogenase 7	Steroid biosynthesis	0.52	0.63	–	–
1423141_at	Lipa	lysosomal acid lipase A	Steroid biosynthesis	–	–	1.84	–
1426913_at	Lss	lanosterol synthase	Steroid biosynthesis	–	–	–	0.61
1417696_at	Soat1	sterol O-acyltransferase 1	Steroid biosynthesis	–	–	1.84	–
1434362_at	Soat1	sterol O-acyltransferase 1	Steroid biosynthesis	–	–	1.56	–
1448068_at	Soat1	sterol O-acyltransferase 1	Steroid biosynthesis	–	1.52	–	–
1460722_at	Soat2	sterol O-acyltransferase 2	Steroid biosynthesis	–	–	–	0.67
1445321_at	Sqle	squalene epoxidase	Steroid biosynthesis	–	–	2.87	–
1424182_at	Acat1	acetyl-Coenzyme A acetyltransferase 1	Synthesis and degradation of ketone bodiesTerpenoid backbone biosynthesis	–	1.56	–	–
1419506_at	Ggps1	geranylgeranyl diphosphate synthase 1	Terpenoid backbone biosynthesis	–	–	1.69	–
1433443_a_at	Hmgcs1	3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1	Synthesis and degradation of ketone bodiesTerpenoid backbone biosynthesis	–	–	1.54	–
1433444_at	Hmgcs1	3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1	Synthesis and degradation of ketone bodiesTerpenoid backbone biosynthesis	0.66	–	–	–
1423804_a_at	Idi1	isopentenyl-diphosphate delta isomerase	Terpenoid backbone biosynthesis	–	1.83	–	–
1451122_at	Idi1	isopentenyl-diphosphate delta isomerase	Terpenoid backbone biosynthesis	–	1.56	–	–
Lipid transport
1422526_at	Acsl1	acyl-CoA synthetase long-chain family member 1	PPAR signaling pathway	–	–	1.51	–
1450643_s_at	Acsl1	acyl-CoA synthetase long-chain family member 1	PPAR signaling pathway	–	–	1.61	–
1447355_at	Acsl1	acyl-CoA synthetase long-chain family member 1	PPAR signaling pathway	–	0.14	–	–
1451828_a_at	Acsl4	acyl-CoA synthetase long-chain family member 4	PPAR signaling pathway	0.35	0.65	–	–
1418911_s_at	Acsl4	acyl-CoA synthetase long-chain family member 4	PPAR signaling pathway	0.52	–	–	–
1433531_at	Acsl4	acyl-CoA synthetase long-chain family member 4	PPAR signaling pathway	0.64	–	–	–
1444231_at	Acsl5	acyl-CoA synthetase long-chain family member 5	PPAR signaling pathway	–	3.08	–	–
1423166_at	Cd36	CD36 antigen	PPAR signaling pathway	–	1.62	4.85	–
1450883_a_at	Cd36	CD36 antigen	PPAR signaling pathway	–	–	4.00	–
1450884_at	Cd36	CD36 antigen	PPAR signaling pathway	–	–	3.33	–
1418438_at	Fabp2	fatty acid binding protein 2, intestinal	PPAR signaling pathway	2.26	2.02	–	–
1417023_a_at	Fabp4	fatty acid binding protein 4, adipocyte	PPAR signaling pathway	–	–	1.62	–
1425809_at	Fabp4	fatty acid binding protein 4, adipocyte	PPAR signaling pathway	–	–	–	1.55
1416021_a_at	EG620603 /// Fabp5	fatty acid binding protein 5, epidermal	PPAR signaling pathway	3.10	–	–	3.49
1416022_at	Fabp5	fatty acid binding protein 5, epidermal	PPAR signaling pathway	2.46	–	–	6.14
1450779_at	Fabp7	fatty acid binding protein 7, brain	PPAR signaling pathway	–	3.08	–	–
1422811_at	Slc27a1	solute carrier family 27 (fatty acid transporter), member 1	PPAR signaling pathway	0.58	–	–	–
1417963_at	Pltp	phospholipid transfer protein	PPAR signaling pathway	–	1.56	1.89	–
1456424_s_at	Pltp	phospholipid transfer protein	PPAR signaling pathway	–	–	2.10	–
1421821_at	Ldlr	low density lipoprotein receptor	Not applicable	–	–	–	0.62
1434465_x_at	Vldlr	very low density lipoprotein receptor	Not applicable	–	–	1.67	–
Fatty acid oxidation
1424853_s_at	Cyp4a10 /// Cyp4a31	cytochrome P450, family 4, subfamily a, polypeptide 10 /// cytochrome P450, family 4, subfamily a, polypeptide 31	PPAR signaling pathway	–	0.60	0.55	–
1424352_at	Cyp4a12a	cytochrome P450, family 4, subfamily a, polypeptide 12a	PPAR signaling pathway	–	–	1.57	–
1423257_at	Cyp4a14	cytochrome P450, family 4, subfamily a, polypeptide 14	PPAR signaling pathway	0.63	0.35	0.33	–
1440134_at	Cyp4a31	cytochrome P450, family 4, subfamily a, polypeptide 31	PPAR signaling pathway	0.59	0.56	0.56	–
1424943_at	Cyp4a31	cytochrome P450, family 4, subfamily a, polypeptide 31	PPAR signaling pathway	0.61	–	–	–
Lipid efflux
1426959_at	Bdh1	3-hydroxybutyrate dehydrogenase, type 1	Synthesis and degradation of ketone bodies	1.58	1.60	–	–
1452257_at	Bdh1	3-hydroxybutyrate dehydrogenase, type 1	Synthesis and degradation of ketone bodies	1.77	1.60	–	–
1436750_a_at	Oxct1	3-oxoacid CoA transferase 1	Synthesis and degradation of ketone bodies	–	1.58	2.06	–
1425771_at	Akr1d1	aldo-keto reductase family 1, member D1	Primary bile acid biosynthesis	–	2.20	–	–
1455100_at	Akr1d1	aldo-keto reductase family 1, member D1	Primary bile acid biosynthesis	–	1.96	2.06	–
1422100_at	Cyp7a1	cytochrome P450, family 7, subfamily a, polypeptide 1	Primary bile acid biosynthesisPPAR signaling pathway	0.23	2.56	–	0.53
1438743_at	Cyp7a1	cytochrome P450, family 7, subfamily a, polypeptide 1	Primary bile acid biosynthesisPPAR signaling pathway	0.32	2.86	–	0.52
1421074_at	Cyp7b1	cytochrome P450, family 7, subfamily b, polypeptide 1	Primary bile acid biosynthesis	–	–	–	1.80
1421075_s_at	Cyp7b1	cytochrome P450, family 7, subfamily b, polypeptide 1	Primary bile acid biosynthesis	–	–	–	1.99
1449309_at	Cyp8b1	cytochrome P450, family 8, subfamily b, polypeptide 1	Primary bile acid biosynthesisPPAR signaling pathway	0.52	0.55	–	–
1417590_at	Cyp27a1	cytochrome P450, family 27, subfamily a, polypeptide 1	Primary bile acid biosynthesisPPAR signaling pathway	0.65	–	0.65	–
1418780_at	Cyp39a1	cytochrome P450, family 39, subfamily a, polypeptide 1	Primary bile acid biosynthesis	0.25	–	0.54	–
1419975_at	Scp2	sterol carrier protein 2, liver	Primary bile acid biosynthesisPPAR signaling pathway	–	–	0.52	–
1421839_at	Abca1	ATP-binding cassette, sub-family A (ABC1), member 1	Not applicable	0.53	–	–	–
1450392_at	Abca1	ATP-binding cassette, sub-family A (ABC1), member 1	Not applicable	0.45	–	–	–
1457554_at	Apob	apolipoprotein B	Not applicable	0.45	–	1.68	–
1419399_at	Mttp	microsomal triglyceride transfer protein	Not applicable	0.56	0.60	–	–
Transcription factor
1449051_at	Ppara	peroxisome proliferator activated receptor alpha	PPAR signaling pathway	–	–	2.18	–
1420715_a_at	Pparg	peroxisome proliferator activated receptor gamma	PPAR signaling pathway	1.61	–	1.85	–
1425762_a_at	Rxra	retinoid X receptor alpha; similar to retinoid X receptor-alpha	PPAR signaling pathway	0.62	–	–	–
1426744_at	Srebf2	sterol regulatory element binding factor 2	Not applicable	0.38	–	1.77	–
1426690_a_at	Srebf1	sterol regulatory element binding transcription factor 1	Not applicable	1.62	2.51	3.35	1.64

-: not changed

A summary of differentially expressed genes related to lipid metabolism is shown in Table 4. Among the genes related to fatty acid biosynthesis, acetyl-coenzyme A carboxylase α (Acaca), Acly, and fatty acid synthase (Fasn), among others, showed increased expression in the nine-week HFD-fed BALB/c group, suggesting that fatty acid biosynthesis may be activated. Other genes related to fatty acid biosynthesis, including Scd1 and malic enzyme (Me), showed decreased expression in both the one- and four-week HFD-fed BALB/c groups, suggesting that fatty acid biosynthesis may be down-regulated in these groups. In the nine-week HFD-fed B6 group, the expression of some genes was decreased; however, Acaca expression was increased.

In TAG biosynthesis, glycerophosphate acyltransferase, lysophosphatidate acyltransferase, and diacylglycerol acyltransferase may act as regulatory enzymes (Nguyen et al. 2007). As shown in Table 4, 1-acylglycerol-3-phosphate O-acyltransferase (Agpat) 2 and Agpat6, diacylglycerol O-acyltransferase 2 (Dgat2), mitochondrial glycerol-3-phosphate acyltransferase (Gpam), and lysocardiolipin acyltransferase 1 (Lclat1) are the genes encoding the abovementioned enzymes. In the nine-week HFD-fed BALB/c group, although the expression of many genes related to glycerolipid metabolism increased, of the genes related to TAG metabolism, only Agpat2 showed increased expression. In both the one- and four-week HFD-fed BALB/c groups, there were both increases and decreases in the expression of genes involved in TAG biosynthesis.

Many genes related to steroid biosynthesis showed increased expression in both the four- and nine-week HFD-fed BALB/c groups. On the other hand, a few genes showed decreased expression in the one-week HFD-fed BALB/c and the nine-week HFD-fed B6 groups.

Among the lipid transport genes, CD36 antigen (Cd36) and solute carrier family 27 member 1 (Sld27a1) are responsible for the transport of fatty acids. Cd36 expression increased in four-week and nine-week HFD-fed BALB/c groups. In addition, the number of probes increased more in the nine-week than in the four-week HFD-fed BALB/c group. Therefore, fatty acid import in the liver was increased in these groups, and may have been more active in the nine-week HFD-fed BALB/c group. Pospholipid transfer protein (Pltp), which is related to reverse cholesterol transport to the liver, was up-regulated in the four-week and nine-week HFD-fed BALB/c groups.

As to the reduction in lipid accumulation in the liver, lipid oxidation or lipid efflux could be related.

The expression of genes related to lipid oxidation was down-regulated in the one-, four-, and nine-week HFD-fed BALB/c groups. However, the gene expression of carnitine palmitoyltransferase I (Cpt1), which transports fatty acids through the inner mitochondrial membrane for beta-oxidation, was not changed.

Expression of genes involved in synthesis and degradation of ketone bodies was increased in the one-, four-, and nine-week HFD-fed BALB/c groups. Genes related to primary bile acid biosynthesis, including Cyp7a1 (the first and rate-limiting enzyme in the bile acid biosynthetic pathway) were down-regulated in the one-week–fed BALB/c group and up-regulated in the four-week–fed BALB/c group. Srebp1, a key transcription factor of lipid biosynthesis, was up-regulated in all groups. In the nine-week fed BALB/c group, PPAR-α, PPAR-γ, and Srebp2 were also up-regulated.

Discussion

In the present study, in order to increase our understanding of the key changes in hepatic steatosis in HFD-fed BALB/c mice identified in our previous study (Nishikawa et al. 2007; Nishikawa et al. 2008), we compared gene expression in the liver between HFD-fed BALB/c mice and B6 mice. Moreover, to understand the metabolic changes in the liver leading up to steatosis, we examined gene expression in BALB/c mice at two different feeding periods (one week and four weeks).

In the HFD-fed groups, peri-epididymal fat weight and serum Cho were increased at all time points examined. However, liver weight increased only in the nine-week HFD-fed BALB/c group. Histopathology showed that lipid accumulation in the liver was severe only in the nine-week HFD-fed BALB/c group. Therefore, lipid accumulation in the liver may occur subsequent to abdominal fat accumulation and the increase in serum Cho.

Metabolic changes predicted from the results of our microarray analysis are shown in Figure 2 . In the nine-week HFD-fed BALB/c group, marked differences from other groups are increased expression of fatty acid biosynthesis–related genes (Acaca, Acly, and Fasn) and increases in fatty acid uptake–related genes (Cd36).

Figure 2.

Predicted lipid metabolic change for each HFD-fed group. In the nine-week HFD-fed BALB/c group, fatty acid uptake and fatty acid biosynthesis may be markedly up-regulated. In the four-week HFD-fed BALB/c group, fatty acid uptake may be up-regulated, but biosynthesis may be down-regulated. In the one-week HFD-fed BALB/c group, fatty acid biosynthesis may be down-regulated. In both the control and the nine-week HFD-fed B6 groups, lipid metabolism may be balanced. Cho, cholesterol; FA, fatty acids; TAG, triacylglycerol; VLDL, very low-density lipoprotein.

In fatty acid biosynthesis, ACACA, FASN, and ACLY are all important. ACACA, which is involved in carboxylation of acetyl-CoA to malonyl-CoA, is the most important enzyme in the regulation of lipogenesis (Botham and Mayes 2006). ACLY catalyzes the cleavage of citrate to oxaloacetate and acetyl-CoA, releasing acetyl-CoA, which can then be used in fatty acid biosynthesis in the extramitochondrial cytosol (Botham and Mayes 2006). In leptin receptor–deficient db/db mice where hepatic ACLY is abnormally elevated, targeted suppression of ACLY in the liver can correct obesity-associated fatty liver (Wang et al. 2009). FASN catalyzes the last step in fatty acid biosynthesis and thus is believed to be a major determinant of the maximal hepatic capacity to generate fatty acids by de novo lipogenesis (Dorn et al. 2010). In twelve-week HFD-fed BALB/c mice with steatosis and in humans with nonalcoholic fatty liver disease, FASN expression in the liver was higher compared to the respective normal control (Dorn et al. 2010). Therefore, induction of ACLY, ACACA, and FASN may be one of the causes of excessive fat accumulation in the livers of the nine-week HFD-fed BALB/c group.

CD36 antigen acts on hepatocytes as a facilitator of long-chain fatty acid transport, but the mechanism by which it facilitates fatty acid uptake remains vague (Silverstein and Febbraio 2009). Fatty acids in hepatocytes may be converted to TAG or oxidized as fuel. When intrahepatic fatty acids are present in excess, the majority of the excess fatty acid is likely to be converted to TAG, which may be stored (Bradbury 2006). In models in which CD36 was specifically induced in the liver by pharmacological means or by cDNA transduction, CD36 contributed to steatosis (Silverstein and Febbraio 2009). In subjects with extreme steatosis, CD36 gene expression is up-regulated (Greco et al. 2008). Considering these reports, the increase of lipid uptake induced by CD36 may contribute to the severe hepatic fatty accumulation seen in the nine-week HFD-fed BALB/c group.

In the nine-week HFD-fed B6 group, a few genes related to lipid metabolism were differentially expressed, suggesting that lipid metabolism may be balanced by the gene expression level at this point.

In both the one- and four-week HFD-fed BALB/c groups, fatty acid biosynthesis was considered to be down-regulated. SCD1 catalyzes the synthesis of monounsaturated long-chain fatty acids from saturated fatty acyl-CoAs. SCD1-deficient mice had decreased liver fat content, suggesting that the lack of SCD1 provided protection from fatty accumulation in the liver (Peter et al. 2011).

An increase in Cd36 expression was also observed in the four-week HFD-fed BALB/c group, although not to the extent observed in the nine-week–fed BALB/c group, suggesting that the increased lipid uptake may occur at least by four weeks and then increase with time. In the one-week HFD-fed BALB/c group, no change was observed.

Several studies examining gene expression in HFD-fed mice have been reported. However, the relationship between histopathology and gene expression is not clear. In a study of HFD-fed B6 mice, several genes involved in lipogenesis (e.g., Acly, Me, and Fasn) were induced and they later returned to baseline levels, whereas genes involved in Cho synthesis (e.g., HMG-CoA reductase, farnesyl pyrophosphate synthetase, squalene synthase) were repressed after eleven days of feeding HFD; no steatosis of the liver was observed at this point (Gregoire et al. 2002). In twelve-week HFD-fed C57BL/6J mice, the expression of genes involved in fatty acid beta-oxidation and ketone body synthesis (e.g., Aox1, acetyl-CoA acyltransferase 1, HMG-CoA lyase) and fatty acid uptake into the liver (Cd36/Fat) was increased, and that of genes involved in lipogenesis (e.g., Fasn, acetyl-CoA synthetase 2, Gpam, Me) and Cho synthesis (squalene epoxidase (Sqle), farnesyl diphosphate farnesyl transferase 1) was decreased (Kim et al. 2004). In twenty-six-week HFD-fed mice, genes involved in fatty acid oxidation (e.g., Cyp4a10, Cyp4a14) were up-regulated, whereas those involved in lipogenesis (e.g., Me, Scd1) were suppressed (Patsouris et al. 2006). In a nine-month study of HFD-fed obese C57BL mice, genes related to beta-oxidation (e.g., carnitine O-acetyltransferase, Cpt1) were up-regulated and those related to Cho biosynthesis (e.g., Cyp7a1, Sqle) were down-regulated. On the other hand, in nine-month HFD-fed lean C57BL mice, genes involved in fatty acid biosynthesis (e.g., Scd1, Me, Fasn) were down-regulated (de Fourmestraux et al. 2004). From these reports, it appears that short-term HFD feeding may lead to induction of lipogenesis, but long-term HFD-feeding may lead to a return to baseline or suppression of fatty acid biosynthesis and Cho synthesis, and induction of beta-oxidation and fatty acid intake. Kim et al. hypothesized that in the twelve-week HFD-fed mice, the uptake of fatty acids into the liver was augmented, resulting in subsequent accumulation of TAG in the liver, and that hepatic TAG accumulation might have driven the up-regulation of genes involved in lipid catabolism and the down-regulation of lipogenic genes by a feedback mechanism. Therefore, down-regulation or no change of lipogenesis in long-term HFD-fed animals is considered to be an adaptive change to excessive lipid intake in the liver, and it can be applied to the one-and four-week HFD-fed BALB/c groups in this study. Moreover, at these time points, the histopathological changes were not so severe. In the nine-week HFD-fed BALB/c group, although the up-regulation of fatty acid uptake corresponded with the above reports, the up-regulation of fatty acid biosynthesis may not occur in the same way as in the short-term HFD induction of lipogenesis and probably indicates the collapse of the lipid metabolic system, inducing marked fat accumulation, as observed morphologically.

In conclusion, the severe fatty accumulation in BALB/c mice may be caused, at least in part, by up-regulated fatty acid uptake and fatty acid biosynthesis in BALB/c mice. Cd36, Acaca, Acly, and Fasn may be involved in these metabolic processes.

Footnotes

Acknowledgments

The authors would like to thank Mr. Naoya Masutomi, Mr. Toshinobu Shimizu, Ms. Natsuko Terasaki, Ms. Manami Miyake, and Mr. Yoshimi Inoue for skillful technical assistance and helpful advice concerning the microarray analysis.

The author(s) declared no potential conflicts of interest with respect to the authorship and/or publication of this article. The author(s) received no financial support for the research and/or authorship of this article.

Abbreviations

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