Abstract
Serum total cholesterol amounts in the stroke-prone hypertensive rat (SHRSP) strain are lower than in the normotensive control strain, Wistar-Kyoto (WKY) rat. To understand the strain difference, constitutive gene expression levels of hepatic cholesterol biosynthetic enzymes in male 8-week-old SHRSP and WKY rats were comparatively examined by DNA microarray and real-time reverse transcription-polymerase chain reaction (RT-PCR) analyses. Of 22 cholesterol biosynthetic enzyme genes, expression levels of 8 genes, Pmvk, Idi1, Fdps, Fdft1, Sqle, Lss, Sc4mol, and Hsd17b7, in SHRSP were less than 50% those of the WKY rats; especially, the expression level of Sqle gene, encoding squalene epoxidase, a rate-limiting enzyme in cholesterol biosynthesis pathway, was about 20%. The gene expression level of sterol regulatory element-binding protein-2 (SREBP-2), which functions as a transcription factor upregulating gene expression of cholesterol biosynthetic enzymes, in SHRSP was about 70% of that in WKY rats. These results demonstrate the possibility that the lower serum total cholesterol level in SHRSP is defined by lower gene expression of most hepatic cholesterol biosynthetic enzymes. In particular, decreased gene expression level of Sqle gene might be the most essential factor. Moreover, the broad range of lowered rates of these genes in SHRSP suggests that the abnormal function and/or expression not only of SREBP-2 but also of one or more other transcription factors for those gene expressions exist in SHRSP.
Stroke-prone spontaneously hypertensive rats (SHRSP) are widely used as a genetic model animal for hypertension and stroke.1,2) This strain also shows significantly lower serum total cholesterol (T-CHO) than the normotensive control strain, Wistar-Kyoto (WKY) rats, when both strains are maintained on a standard diet.3–5) However, the high-fat and high-cholesterol diet-mediated development of hypercholesterolemia occurs more efficiently in SHRSP than in WKY rats.6–8) These findings suggest that there are differences in genetic factors responsible for cholesterol homeostasis.
Serum T-CHO amount is primarily regulated by cholesterol biosynthesis, metabolism of cholesterol to bile acids, and uptake of cholesterol into cells via low-density lipoprotein (LDL) receptor in the liver. It is therefore one of important subjects for understanding the mechanism of strain difference between SHRSP and WKY strains, maintained on a standard diet, in serum T-CHO amount to look into strain differences in the expression and/or function of hepatic enzymes involved in cholesterol biosynthesis (Fig. 1). As for such differences, only two enzymes, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and mevalonate pyrophosphate decarboxylase (MVD), have been reported.9–12) Since HMGCR activity was higher in SHRSP than in WKY rats,9) HMGCR is hardly thought to be a cause of the lower serum T-CHO level in SHRSP. On the other hand, the expression and activity levels of MVD were lower in SHRSP,10–12) suggesting that MVD might be a cause of the lower serum T-CHO level.
To further understand the difference between SRHSP and WKY rats maintained on a standard diet in serum T-CHO, we comparatively examined the expression levels of the genes responsible for the biosynthesis of cholesterol together with that of the gene of sterol regulatory element-binding protein-2 (SREBP-2), a common transcription factor of the cholesterol biosynthetic enzyme genes.13,14) The results obtained herein indicated that the expression levels of most of the genes tested were significantly lower in SHRSP than in WKY rats.
Materials and Methods
AnimalsMale WKY/Izm and SHRSP/Izm strains were supplied by the Disease Model Cooperative Research Association, Japan, and were used at 8 weeks of age. The rats were kept in plastic cages in an air-conditioned room with a 12-h light/12-h dark cycle, and given a standard diet, MF (Oriental Yeast, Tokyo, Japan), and water ad libitum. All rats used in the present experiments were sacrificed by decapitation. The liver of each rat was removed, cut into small pieces, and then frozen and stored at −80°C until use. The experimental protocols were approved by the Animal Experimentation Ethic Committee at the University of Shizuoka.
Microarray AnalysisTotal RNA was extracted from each frozen rat liver (approximately 100 mg) using TriPure reagent (Roche Applied Science, Indianapolis, IN, U.S.A.) in accordance with the manufacturer’s instructions. Total RNA (500 ng), which included equal amounts of total RNAs derived from individual four rats in each strain, was labeled with Cyanine-3 using a Quick-Amp Labeling Kit (Agilent Technologies, Palo Alto, CA, U.S.A.). Fluorescently labeled targets were hybridized to Whole Rat 4×44 K oligo DNA microarrays (Agilent Technologies). Hybridization and washing processes were performed according to the manufacturer’s instructions, and hybridized microarrays were scanned using an Agilent Microarray Scanner (Agilent Technologies). Feature extraction software (Agilent Technologies) was employed for the image analysis and data extraction processes. The raw array data were normalized using Subio Platform (Subio, Tokyo, Japan) software.
Reverse Transcription-Polymerase Chain Reaction (RT-PCR)The total RNAs prepared from individual four rats in each strain of rats were applied to real-time RT-PCR to determine the expression levels of the genes for cholesterol biosynthetic enzymes, Srebf2 and glyceraldehyde-3-phosphate-dehydrogenase (Gapdh). A portion (8 µg) of the total RNA was converted into cDNA using polyd(N)6 random primer (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, U.K.) and Moloney murine leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA, U.S.A.) in an RT reaction mixture (250 µL). The reaction was performed with a GeneAmp with SYBR Green PCR Core Reagents (Applied Biosystems) in 25 µL of total reaction mixture containing 5 µL of the RT reaction mixture, 0.5 µM of each primer (forward and reverse primers), and AmpliTaq Gold DNA polymerase (Applied Biosystems). The amplification protocol consisted of 40 cycles of denaturation, annealing, and extension. The level of each cDNA was assessed by the relative standard curve method, as described in the PE Applied Biosystems User Bulletin 2, 1997, and normalized to the level of the Gapdh gene. The primer sequences and conditions for PCR (denaturation, annealing, and extension) are listed in Table 1.
Table 1. Nucleotide Sequences of Primers and Reaction Conditions for Real-Time RT-PCR
| Gene | Nucleotide sequences of primers | Reaction condition |
|---|
| Denaturation | Annealing | Elongation |
|---|
| Gapdh | 5′TGT GAA CGG ATT TGG CCG TA-3′ (forward) | 95°C, 1 min | 60°C, 1 min | 72°C, 2 min |
| 5′-TCG CTC CTG GAA GAT GGT GA-3′ (reverse) | | | |
| Hmgcr | 5′-CCA GGA TGC AGC ACA GAA TGT -3′ (forward) | 95°C, 1 min | 60°C, 1 min | 72°C, 2 min |
| 5′-CCA ATT CGG GCA AGC TGC CG-3′ (reverse) | | | |
| Pmvk | 5′-TAG TAG CCT CGG AGC AGA GTC-3′ (forward) | 95°C, 30 s | 62°C, 30 s | 72°C, 1 min |
| 5′-CCT CCA GGC ACT GTT CAT CTC-3′ (reverse) | | | |
| Mvd | 5′-CTG CTG CGA ATG GAG ACA AG-3′ (forward) | 95°C, 30 s | 60°C, 30 s | 72°C, 1 min |
| 5′-GCT CCG TAG CCA GCG AAG T-3′ (reverse) | | | |
| Idi1 | 5′-GGA TAC CCT TGG AAG AGG TTG A-3′ (forward) | 95°C, 30 s | 62°C, 30 s | 72°C, 1 min |
| 5′-TCC GGA TTC AGG GTT ACA TTC T-3′ (reverse) | | | |
| Fdps | 5′-TGG TGT GTA GAA CTG CTC CAG GCT TTC TTC-3′ (forward) | 95°C, 30 s | 65°C, 30 s | 72°C, 1 min |
| 5′-ACA CTG GGG TCT CCA AAG AGA TCA AGG TAG-3′ (reverse) | | | |
| Fdft1 | 5′-GTT ATC CAG GCG CTG GAT GG-3′ (forward) | 95°C, 30 s | 62°C, 30 s | 72°C, 1 min |
| 5′-TGG TCT TCC AGA TAG TCA CG-3′ (reverse) | | | |
| Sqle | 5′-TCA TTT CTG GAG GCC TCT CAG AAT G-3′ (forward) | 94°C, 30 s | 53°C, 30 s | 72°C, 1 min |
| 5′-CCT TTC AGT GAA CCA GAT ACT TCA T-3′ (reverse) | | | |
| Lss | 5′-TGA TGG CTG TCA GGC ATC C-3′ (forward) | 95°C, 30 s | 62°C, 30 s | 72°C, 1 min |
| 5′-GTG TAG CTG ATG GCA CAG GAC TT-3′ (reverse) | | | |
| Cyp51 | 5′-GAT GCT CAT CGG ACT GCT G-3′ (forward) | 95°C, 30 s | 62°C, 30 s | 72°C, 1 min |
| 5′-ATA AAC GAA GCA TAG TGG ACC-3′ (reverse) | | | |
| Sc4mol | 5′-AGA CTC CTT CAC CAC AAG AG-3′ (forward) | 95°C, 30 s | 61°C, 30 s | 72°C, 1 min |
| 5′-CCA GTC CCA AGA ATT AGG GT-3′ (reverse) | | | |
| Hsd17b7 | 5′-GAA CGC CGG AAT CAT GCC TAA CC-3′ (forward) | 95°C, 30 s | 63°C, 30 s | 72°C, 1 min |
| 5′-GGA AAA GCC ACA CCA ATG CCT CTG-3′ (reverse) | | | |
| Srebf2 | 5′-GGA AGG CCA TTG ATT ACA TCA AG-3′ (forward) | 95°C, 30 s | 62°C, 30 s | 72°C, 1 min |
| 5′-TTG CCA GCT TCA GCA CCA T-3′ (reverse) | | | |
Values are expressed as the means±S.D. The statistical significance of differences between the two groups was assessed using the two-tailed Student’s t-test.
Results
The hepatic expression levels of all genes encoding cholesterol biosynthetic enzymes in the livers of SHRSP and WKY rats maintained on a standard diet MF were analyzed by a DNA microarray technique. As shown in Table 2, the expression levels of most of the genes in SHRSP were lower than in WKY rat. Among them, the levels of Pmvk, Idi1, Fdps, Fdft1, Sqle, Lss, Tm7sf2, Sc4mol, and Hsd17b7 mRNAs in SHRSP were less than 50% of those in WKY rats.
Table 2. Expression Levels of Hepatic mRNAs for Cholesterol Biosynthetic Enzymes or SREBP-2 in SHRSP as Compared with Those in WKY Rats
| Feature No. | Gene | Protein | Expression level (ratio to WKY) |
|---|
| Microarray | Real-time RT-PCR |
|---|
| 6855 | Acat1 | Acetyl-CoA C-acetyltransferase | 0.98 | |
| 38770 | Acat1 | 0.96 | |
| 26918 | Acat2 | 0.45 | |
| 840 | Hmgcs1 | Hydroxymethylglutaryl-CoA synthase | 0.66 | |
| 18194 | Hmgcs1 | 0.65 | |
| 23546 | Hmgcs2 | 0.77 | |
| 9081 | Hmgcr | 3-Hydroxy-3-methylglutaryl-CoA reductase | 1.47 | 0.79±0.19 |
| 23491 | Mvk | Mevalonate kinase | 0.60 | |
| 28017 | Pmvk | Phosphomevalonate kinase | 0.47 | 0.49±0.062*** |
| 38688 | Mvd | Mevalonate pyrophosphate decarboxylase | 0.62 | 0.64±0.14* |
| 18852 | Idi1 | Isopentenyl-diphosphate δ-isomerase | 0.44 | 0.36±0.088** |
| 38809 | Idi1 | 0.33 |
| 9588 | Ggps1 | Geranylgeranyl diphosphate synthase 1 | 1.24 | |
| 8001 | Fdps | Farnesyl diphosphate synthase (farnesyl pyrophosphate synthetase, dimethylallyltranstransferase, geranyltranstransferase) | 0.42 | 0.39±0.020*** |
| 20433 | Fdps | 0.45 |
| 36906 | Fdps | 1.52 |
| 18144 | Fdft1 | Farnesyl-diphosphate farnesyltransferase (squalene synthase) | 0.45 | 0.50±0.23* |
| 37588 | Sqle | Squalene epoxidase | 0.20 | 0.21±0.032*** |
| 40868 | Lss | Lanosterol synthase | 0.46 | 0.39±0.094*** |
| 30380 | Cyp51 | Cyp51 (sterol 14-demethylase) | 0.52 | 0.54±0.17* |
| 25526 | Tm7sf2 | Δ-Sterol reductase | 0.44 | |
| 20848 | Sc4mol | Methylsterol monooxygenase | 0.26 | 0.28±0.026*** |
| 1126 | Nsdhl | Sterol-4α-carboxylate 3-dehydrogenase | 0.76 | |
| 43331 | Hsd17b7 | Hydroxysteroid (17β) dehydrogenase 7 | 0.24 | 0.34±0.093*** |
| 727 | Ebp | Emopamil binding protein (sterol isomerase) | 0.77 | |
| 31546 | Dhcr24 | 24-Dehydrocholesterol reductase | 0.90 | |
| 36152 | Dhcr7 | 7-Dehydrocholesterol reductase | 0.84 | |
| 6611 | Lipa | Lipase A, lysosomal acid, cholesterol esterase | 0.88 | |
| 33689 | Soat1 | Sterol O-acyltransferase | 0.78 | |
| 22671 | Soat2 | 0.95 | |
| 17383 | Sc5dl | Sterol-C5-desaturase | 0.74 | |
| 9895 | Srebf2 | Sterol regulatory element-binding protein-2 (SREBP-2) | 0.58 | 0.73±0.13* |
| 40025 | Srebf2 | 0.61 |
Expression level represents ratio of hepatic mRNA levels in SHRSP to those in WKY rats. In microarray analysis, total RNA sample including equal amounts of total RNAs derived from individual four rats in each strain was used. In real-time RT-PCR analysis, the mRNA levels were determined using Gapdh gene as an internal control, and the ratio of each mRNA level in SHRSP to the corresponding mRNA level in WKY rats is shown as mean±S.D. (n=4/strain). *, **, *** Statistically significant differences from the corresponding mRNA levels in WKY rats: * p<0.05, ** p<0.01, *** p<0.001. “Feature No.” was obtained from the data sheet from Whole Rat 4×44 K oligo DNA microarray (Agilent Technologies).
Of the 9 genes named above, the expression levels of Pmvk, Idi1, Fdps, Fdft1, Sqle, Lss, Sc4mol, and Hsd17b7 genes along with Hmgcr, Mvd, and Cyp51 genes, whose expression levels in SHRSP were not less than 50% of those in WKY rats in DNA microarray analysis, were further estimated by real-time RT-PCR (Table 2). The results showed that the levels of Pmvk, Mvd, Idi1, Fdps, Fdft1, Sqle, Lss, Cyp51, Sc4mol, and Hsd17b7 mRNAs were significantly lower in SHRSP than in WKY rats. Moreover, Sqle gene expression had the lowest level (about 20% of the WKY rat level) among all genes tested. In addition, no significant difference was found between SHRSP and WKY rats in the expression level of Hmgcr mRNA.
The expression level of the Srebf2 gene encoding sterol regulatory element-binding protein-2 (SREBP-2), which functions as a transcription factor upregulating the gene expression of cholesterol biosynthetic enzymes,13,14) was also assessed. The real-time RT-PCR analysis showed that its level in SHRSP was about 70% of that in WKY rats, with a statistically significant difference.
Discussion
The present study demonstrated that the hepatic expression levels of most genes for cholesterol biosynthetic enzymes, including Pmvk, Mvd, Idi1, Fdps, Fdft1, Sqle, Lss, Cyp51, Sc4mol, and Hsd17b7 genes were lower in SHRSP maintained on a standard diet than in WKY rats. Those results therefore suggested that the lower serum T-CHO levels in SHRSP than in WKY rats depend on the low efficiency of cholesterol biosynthesis in the liver of SHRSP resulting from the reduced expression of those hepatic genes but not of a particular gene.
Until now, the hepatic cholesterol biosynthetic enzymes, whose protein activity and/or expression has been compared between SHRSP and WKY rats, included only two enzymes: HMGCR and MVD.9–12) The activity of HMGCR in SHRSP has been reported to be higher than that in WKY rats.9) DNA microarray analysis in our present study showed that the level of Hmgcr gene expression in SHRSP was about 1.5-times higher than in WKY rats, while in real-time RT-PCR analysis no such significant difference could be shown. Considering from the previous report9) and present findings, the quantitative and qualitative differences of hepatic HMGCR between SHRSP and WKY rats would not be main causes of strain difference (SHRSP<WKY) in serum T-CHO level. As for MVD, Michihara et al. reported that the levels of its activity, amount, and gene expression were lower in SHRSP than in WKY rats.10–12) In the present study, such strain difference in the gene expression level was also shown. However, judging from the lower expression levels our results showed for many of the genes for the other cholesterol biosynthetic enzymes in SHRSP, the expression level of the Mvd gene cannot necessarily define the lower serum T-CHO level in SHRSP.
HMGCR is the rate-limiting enzyme in the cholesterol biosynthesis pathway. However, we here supposed that its expression level is not likely to have a substantial effect on the lower level of cholesterol biosynthesis in the SHRSP liver. The present study showed that strain difference (SHRSP<WKY) in hepatic gene expression level of SQLE (squalene epoxidase), which is also one of the rate-limiting enzymes in the cholesterol biosynthesis pathway,15–17) was the largest among the genes for cholesterol biosynthetic enzymes. Therefore, the SQLE expression level might be the most responsible for the lower serum T-CHO levels in SHRSP.
Furthermore, the present study showed that the expression level of the hepatic Srebf2 gene in SHRSP was about 70% of that in WKY rats. In the light of the function of SREBP-2 in positively controlling the expression of genes for cholesterol biosynthetic enzymes in common,13,14) the downregulation of Srebf2 gene expression is presumed to uniformly lead to the reduced expression of these genes. However, the reduced expression rates of the genes for cholesterol biosynthetic enzymes in SHRSP relative to those in WKY rats varied. For example, the level of Hmgcr mRNA in SHRSP was similar to that in WKY rats in real-time RT-PCR analysis, whereas Sqle mRNA in SHRSP was about 20% of that in WKY rats in DNA microarray and real-time RT-PCR analyses. Such diversity suggests that decreased SREBP-2 expression cannot necessarily be a causal factor in the reduced expression of these genes. In other words, some sort of transcription factors controlling the expression of these genes in addition to SREBP-2 must have abnormal functions in SHRSP.
The present data suggest that lower constitutive hepatic expression levels of the genes for cholesterol biosynthetic enzymes, especially SQLE, in SHRSP can give rise to the lower serum T-CHO levels in SHRSP as compared with those in WKY rats. Further studies on abnormal functions of SREBP-2 and other transcription factors for the genes encoding cholesterol biosynthetic enzymes in SHRSP are necessary to clarify the precise mechanism underlying the strain difference in constitutive serum T-CHO level. Furthermore, metabolism of cholesterol to bile acids and uptake of cholesterol into cells via LDL receptor in the liver are also important for regulation of serum T-CHO amount, so evaluation of gene and protein expressions responsible for those pathways should be an issue in the future.
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