Lower Squalene Epoxidase and Higher Scavenger Receptor Class B Type 1 Protein Levels Are Involved in Reduced Serum Cholesterol Levels in Stroke-Prone Spontaneously Hypertensive Rats

Abstract

A lower serum cholesterol level was recently shown to be one of the causes of stroke in an epidemiological study. Spontaneously hypertensive rats stroke-prone (SHRSP) have lower serum cholesterol levels than normotensive Wistar-Kyoto rats (WKY). To elucidate the mechanisms responsible for the lower serum cholesterol levels in SHRSP, we determined whether the amounts of cholesterol biosynthetic enzymes or the receptor and transporter involved in cholesterol uptake and efflux in the liver were altered in SHRSP. When the mRNA levels of seven cholesterol biosynthetic enzymes were measured using real-time polymerase chain reaction (PCR), farnesyl pyrophosphate synthase and squalene epoxidase (SQE) levels in the liver of SHRSP were significantly lower than those in WKY. SQE protein levels were significantly reduced in tissues other than the brain of SHRSP. No significant differences were observed in low-density lipoprotein (LDL) receptor (uptake of serum LDL-cholesterol) or ATP-binding cassette transporter A1 (efflux of cholesterol from the liver/formation of high-density lipoprotein (HDL)) protein levels in the liver and testis between SHRSP and WKY, whereas scavenger receptor class B type 1 (SRB1: uptake of serum HDL-cholesterol) protein levels were higher in the livers of SHRSP. These results indicated that the lower protein levels of SQE and higher protein levels of SRB1 in the liver were involved in the reduced serum cholesterol levels in SHRSP.

An epidemiological study identified lower serum cholesterol levels as one of the causes of cerebral hemorrhage because the risk of death from cerebral hemorrhage was found to be three-fold higher in men with lower serum cholesterol levels than in those with higher cholesterol levels.^1,2)

Cholesterol is a major constituent of cellular membranes; therefore, reductions in the cholesterol content in the cell have been shown to reduce the proliferation of cells, enhance cell membrane fluidity, and lead to fragile plasma membranes.^3–5) Therefore, lower serum cholesterol is regarded as an important cause of cerebral hemorrhage.

Spontaneously hypertensive rats (stroke-prone) (SHRSP) are rats with severe hypertension and stroke.^6,7) Serum cholesterol levels in these rats are lower than those in normotensive Wistar Kyoto rats (WKY).⁸⁾ Shiota et al. reported that the pathogenesis of stroke in SHRSP was inhibited by the feeding of high fat and cholesterol diets.⁹⁾ These findings showed that reductions in the content of cholesterol in vascular endothelial cells (VECs) in the SHRSP brain due to lower serum cholesterol levels may lead to cerebral hemorrhage following a disruption in the blood–brain barrier (BBB). Disruptions in the BBB have previously been reported in the hypothalamus and hippocampus of SHRSP.^10,11) Furthermore, damage to endothelial cells has been implicated in disruptions in the BBB. Therefore, lower serum cholesterol levels in SHRSP may play a role in the development of cerebral strokes in these rats. The mechanism underlying the disruption of BBB in the brain of SHRSP was also considered as an important cause of stroke.

In order to determine the mechanisms responsible for reducing serum cholesterol levels in SHRSP, the following were examined: 1) absorption of dietary cholesterol (SHRSP>WKY),¹²⁾ 2) disappearance of labeled cholesterol from the serum (SHRSP<WKY),¹³⁾ 3) biosynthesis of cholesterol from acetic acid in the liver (SHRSP<WKY),¹⁴⁾ 4) the activity of cholesterol 7α-hydroxylase (SHRSP=WKY),¹⁵⁾ the rate-limiting enzyme in the formation of bile acid from cholesterol, and 5) amount of plant sterols (SHRSP=WKY) in the serum (12–15%) of rats fed commercial pellets and that in the serum (31–34 mg/dL) and livers (approximately 29 mg/g) of rats fed a 0.5% plant sterol diet,¹⁶⁾ phytosterols compete with cholesterol for intestinal absorption to limit absorption and inhibit the activity of the rate-limiting enzyme of cholesterol biosynthesis (hydroxymethylglutaryl-CoA (HMG-CoA) reductase) and cholesterol 7α-hydroxylase.¹⁷⁾ These findings strongly suggested that the low serum cholesterol levels in SHRSP were caused by a reduction in the hepatic biosynthesis of cholesterol. Certainly, statin has been shown to decrease serum cholesterol levels by accelerating the uptake of low-density lipoprotein (LDL) from serum to the liver, which, in turn, supplements low cholesterol levels in the liver caused by the decreased biosynthesis of cholesterol due to the suppression of HMG-CoA reductase.¹⁸⁾ Lower serum cholesterol levels are considered to be highly influenced by reductions in the content of cholesterol due to the inhibition of cholesterol biosynthetic enzymes in the liver.

The hepatic biosynthesis of cholesterol from acetic acid requires more than 20 enzymatic reactions (Fig. 1). The activity of mevalonate pyrophosphate decarboxylase (MPD), which is involved in cholesterol biosynthesis, was previously shown to be reduced in the livers of SHRSP, while the activity of HMG-CoA reductase, mevalonate kinase (MVK), and phosphomevalonate kinase (PMVK) were similar to those in WKY.¹⁹⁾ We previously demonstrated that the reduced activity of MPD was caused by lower protein levels of MPD in the liver; however, no significant difference was observed in MPD-specific activity between SHRSP and WKY.²⁰⁾ We also reported that MPD mRNA levels in the liver were lower in SHRSP than in WKY.²¹⁾ Furthermore, we found that reductions in MPD expression levels in the livers of SHRSP were suggested to be controlled by the upregulation of microRNA (miR)-214 and downregulation of transcriptional levels.²²⁾ miR was shown to reduce protein levels by inhibiting its synthesis from mRNA or accelerating the degradation of mRNA.^23,24) Recently, it was reported that phosphomevalonate kinase (PMVK), isopentenyl pyrophosphate isomerase (IPI), farnesyl pyrophosphate synthase (FPS), squalene synthase (SQS), squalene epoxidase (SQE), lanosterol synthase (LSS), methylsterol monooxygenase (Sc4mol) and hydroxysteroid (17β) dehydrogenase 7 (Hsd17b7) in the liver of SHRSP were significantly less than 50% those in WKY, when the mRNA levels of 22 cholesterol biosynthetic enzymes measured by real-time polymerase chain reaction (PCR).²⁵⁾ The lower mRNA levels of sterol regulatory element-binding protein 2 (SREBP-2), which is a transcriptional factor regulating gene expression of cholesterol biosynthetic enzymes, also was showed in SHRSP.²⁵⁾ Although PMVK mRNA levels were reduced in the livers of SHRSP, its activity was found to be the same in SHRSP and WKY.^19,25) Furthermore, the mRNA levels of acetoacetyl-CoA transferase, HMG-CoA synthase, HMG-CoA reductase, and MVK in SHRSP were not significantly lower than those in WKY.²⁵⁾ Therefore, the cholesterol biosynthetic enzymes involved in the biosynthesis of mevalonate pyrophosphate from acetyl-CoA were not considered to cause reductions in cholesterol. We previously reported that MPD levels were significantly lower in the brains as well as liver of SHRSP, when the tissue distribution of MPD was examined in WKY and SHRSP.²⁶⁾ Although MPD protein levels were lower in the livers and brains of SHRSP than in those of WKY, it remains unclear whether cholesterol synthase downstream of MPD in SHRSP is reduced. Furthermore, it has not yet been determined whether an increase in the low-density lipoprotein (LDL) receptor (LDLR: participated in the uptake of LDL-cholesterol from the serum to the liver),²⁷⁾ an increase in scavenger receptor class B type 1 (SRB1: participates in the uptake of high-density lipoprotein (HDL)-cholesterol from the serum to the liver),²⁷⁾ or a decrease in ATP-binding cassette transporter A1 (Abca1: involved in the efflux of cholesterol from the liver to the serum/formation of HDL)²⁸⁾ in the livers of SHRSP participates in reducing serum cholesterol contents.

Fig. 1. Cholesterol Biosynthetic Pathway

P: phosphate, PP: pyrophosphate

In the present study, we carried out the comparison of the mRNA levels of the cholesterol biosynthetic enzymes (IPI, FPS, SQS, SQE, LSS showed the reduction of mRNA levels in the liver of SHRSP as described previously) using liver between SHRSP and WKY. Next, we performed the comparison of the protein levels of the enzymes showed the reduction of mRNA levels. Furthermore, we examined the protein levels of LDLR, SRB1, and Abca1 in the liver of SHRSP. The results obtained suggested that the lower protein levels of SQE as well as the higher protein levels of SRB1 played a role in reducing serum cholesterol levels in SHRSP.

MATERIALS AND METHODS

Materials

The ECL Western blotting detection kit was purchased from Amersham Pharmacia Biotech (Tokyo, Japan), BioMasher^® from Nippi, Inc. (Tokyo, Japan), Quick Gene RNA tissue kit S II from FUJIFILM (Tokyo, Japan), SYBR Ex Script RT-PCR kit from TaKaRa (Tokyo, Japan), mouse anti-Farnesyl pyrophosphate synthase immunoglobulin G (IgG), rabbit anti-LDLR IgG, and rabbit anti-Abca1 IgG from Abcam (Tokyo, Japan), goat anti-squalene epoxidase (SQE) (S-17) IgG, goat anti-SRB1 IgG, and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) IgG from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, U.S.A.), rabbit anti-goat IgG conjugated to horseradish peroxidase (HRP) from American Qualex International, Inc. (San Clemente, CA, U.S.A.), goat anti-mouse IgG or goat anti-rabbit IgG conjugated to HRP from Invitrogen Corporation (Carlsbad, CA, U.S.A.), the cholesterol E-test Wako from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), the HDL and very low-density lipoprotein (VLDL)/LDL Quantification Kit from BioVision, Inc., and [¹⁴C]isopentenyl pyrophosphate triammonium salt from Du Pont-New England Nuclear. All other chemicals were of reagent grade, and were purchased from various commercial sources.

Animals

Inbred male (10 weeks old) SHRSP/Izm (average body weight: 220 g) and WKY/Izm (average body weight: 250 g) were obtained from the Disease Model Co-operative Research Association, Japan. Rats were fed pelleted rat food (Lab H Standard; Nosan Corporation Life-Tech Department) ad libitum. All rats were sacrificed at 9:00 for 3 d after purchasing. The experimental protocol was reviewed and approved by the Animal Care and Use Committee of Fukuyama University and complied with guidelines for the care and use of animals by the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Protein Assay

Protein levels were measured by the method of Lowry et al. using bovine serum albumin (BSA) as the standard.²⁹⁾

Cholesterol Synthesis from [¹⁴C]Isopentenyl Pyrophosphate

This experiment was performed as described by Sawamura et al.¹⁹⁾ with minor modifications. Liver slices (0.3 g) were homogenized in three volumes of Krebs–Henseleit solution. After centrifugation at 10000×g for 30 min, 10 mg of the supernatant (S₁₀) was added to cofactor solution A containing 10 mM reduced nicotinamide adenine dinucleotide phosphate (NADPH), 50 mM MgCl₂, 50 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µM leupeptin, and 10 µM pepstatin A. Cofactor solution A was then aerated with a gas mixture of 95% O₂ and 5% CO₂ at 4°C for 10 min. Cofactor solution A containing S₁₀ was incubated at 37°C for the indicated time, after 1 mM [¹⁴C]isopentenyl pyrophosphate (1 µCi) was added. One milliliter of cofactor solution A was subjected to lipid extraction as described by Bligh and Dyer.³⁰⁾ Eighty percent of the chloroform phase was removed and analyzed with TLC (petroleum ether–diethyl ether–acetic acid, 70 : 30 : 4).

2,3-Oxidosqualene Synthesis from [¹⁴C]Isopentenyl Pyrophosphate

This experiment was performed as described by Yamamoto and Block³¹⁾ with minor modifications. Liver slices (0.3 g) were homogenized in three volumes of 100 mM Tris–HCl buffer (pH 7.5) containing 50 mM MgCl₂ and 1 mM FAD. After centrifugation at 10000×g for 30 min, 10 mg of the supernatant (S₁₀) was added to cofactor solution B containing 1 mM [¹⁴C]isopentenyl pyrophosphate (0.5 µCi), 1 mM NADPH, and 0.3% TritonX-100. Cofactor solution B containing S₁₀ was then incubated at 37°C for 1 h. After this incubation, lipids were extracted from 0.6 mL of cofactor solution B. Non-saponifiable fractions were analyzed with TLC (ethylacetate–benzene, 0.5 : 99.5).

Real-Time PCR

Rat tissues (15 mg) were homogenized with a Biomasher (tissue homogenizer; Asist, Tokyo, Japan). After the homogenate was dissolved with appropriate reagents from the Quick Gene RNA tissue kit S II (FUJIFILM, Tokyo, Japan), total RNA (17 µg) was isolated from the homogenate using the Quick Gene RNA tissue kit S II (total RNA extraction kit) and Gene-810 (Nucleic Acid Isolation System; FUJIFILM). The concentration of total RNA was calculated using a Qubit™ fluorometer (Invitrogen). Two-microgram samples of total RNA from each group of tissues were subjected to reverse transcription (RT) using reverse transcriptase in a 50 µL reaction volume. After the RT reaction, the cDNA template was amplified by polymerase chain reaction (PCR) with the SYBR Ex Script RT-PCR kit. SYBR Green was used for the real-time PCR analysis of cholesterol biosynthetic enzymes, which was performed using the ABI7500 system (Applied Biosystems Japan, Tokyo, Japan). Relative gene expression was quantified using GAPDH as an internal control. Primer pairs (listed in Table 1) were designed using the primer Select program of TaKaRa. The DNA products generated by real-time PCR were electrophoresed on a 2% agarose gel, visualized by ethidium bromide staining under UV light, and DNA products were identified as a single band.

Table 1. Real-Time PCR Primer Sequences

Genes	Primer sequence	Size (bases)	Position	Gene Bank
GAPDH	F: 5′-GGCACAGTCAAGGCTGAGAATG-3′	143	242–384	NM_017008
	R: 5′-ATGGTGGTGAAGACGCCAGTA-3′
IPI	F: 5′-GCACTGGCAGGAGTGATTGG-3′	179	233–411	NM_053539
	R: 5′-AGATTGCTGGCATTGATTTCAGG-3′
FPS	F: 5′-AGAATCCGCGTTGAAGCACA-3′	149	115–263	NM_031840
	R: 5′-GGTGTCCCAGTTCATCCTCAGTC-3′
SQS	F: 5′-GCCCTGTCGTTAGCCCTTGA-3′	131	2301–2431	NM_019238
	R: 5′-TTGCAGGCCAGTCTGAGCTAGTTA-3′
SQE	F: 5′-CCCACTTCGTTGGCTTCATT-3′	91	976–1066	NM_017136
	R: 5′-ATGAGAACTGGACTGGGATCGA-3′
LSS	F: 5′-GACTAAGCGTGGCGGGTATTTG-3′	104	1556–1659	NM_031049
	R: 5′-GCATCACTGCGGAGGTACACTCTA-3′
SC5D	F: 5′-TAACGGGTCGGCTCATCACA-3′	90	793–883	NM_053642
	R: 5′-GAGCCTCCGATTCTATCCCACA-3′
DHCR7	F: 5′-ATTGCGTGTGGCTGCCCTA-3′	143	1362–1504	NM_022389
	R: 5′-CTGATGGTTGGTCATTCGGAAG-3′
SREBP-2	F: 5′-GGAAGGCCATTGATTACATCAAG-3′	81	1228–1308	NM_001033694
	R: 5′-TTGCCAGCTTCAGCACCAT-3′
LDLR	F: 5′-CACAACGTCACGCAGCCTAGA-3′	50	2119–2168	NM_175762
	R: 5′-AGAACCGTTGCCTCACACCAG-3′
SRB1	F: 5′-GTTCCGTGAAGATGCAGCTGAG-3′	97	1353–1449	NM_031541
	R: 5′-AACCACAGCAATGGCAGGACTAC-3′
Abca1	F: 5′-AGCAGTTTGTGGCCCTCTTG-3′	157	3800–3956	NM_ 178095
	R: 5′-AGTTCCAGGTTGGGGTACTT-3′

Preparation of the Crude Extract and Immunoblot Analysis

Rat tissues (15 mg) were homogenized with five strokes in a Teflon homogenizer in three volumes of homogenization buffer (50 mM Tris–HCl, pH 7.5 containing 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM 2-mercaptoethanol, 1 mM ethylenediaminetetraacetic acid (EDTA), and protease inhibitors [1 µM leupeptin, 1 µM pepstatin A, 1 µM chymostatin, and 1 µM antipain]) containing 1% Triton X-100. After centrifugation for 1 h at 106000×g, the supernatant was used for immunoblot analysis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on 10% slab gels according to the method of Laemmli.³²⁾ Proteins on the SDS-slab gel were transferred to a nylon membrane (NEN) by electrophoresis, using a modified version of the procedure of Towbin et al.³³⁾ 1) Immunoblot analysis of FPS: FPS on the nylon membrane was detected by incubating with the mouse anti-FPS antibody, washing five times, and then with goat anti-mouse IgG conjugated to HRP. The membrane was washed five times more and was then incubated with rabbit anti-goat IgG conjugated to HRP. 2) Immunoblot analysis of SQE and SRB1 : SQE and SRB1 on the nylon membrane were detected by incubating with the goat anti-SQE antibody or goat anti-SRB1 antibody, washing five times, and then incubating with rabbit anti-goat IgG conjugated to HRP. 3) An immunoblot analysis of LDLR, Abca1, and GAPDH : LDLR, Abca1, and GAPDH on the nylon membrane was performed by incubating with the rabbit anti-LDLR antibody, rabbit anti-Abca1 antibody, or rabbit anti-GAPDH antibody, washing five times, and then incubating with goat anti-rabbit IgG conjugated to HRP. Positive bands were visualized using the ECL Western blotting detection kit (Amersham Pharmacia, Amersham, U.K.), which contained a sensitive chemiluminescent substrate for HRP. Bands were quantified using the Intelligent Quantifier (Bio Image Systems, Inc.). Relative protein levels were quantified using GAPDH as an internal control.

Cholesterol Levels in Tissues

One hundred milligrams in the testis were homogenized in 500 mL of homogenization buffer and centrifuged at 1000×g for 10 min. Forty microliters of postnuclear supernatant (PNS) was mixed with 5 mL of Folch extract (chloroform–methanol, 2 : 1), and the mixture was incubated for 10 min at 37°C with shaking. After the mixture was centrifuged at 3000×g for 10 min, 3 mL of the supernatant was evaporated dry by boiling at 100°C, and then dissolved in 200 µL of isopropyl alcohol containing 1% Triton-X-100. The cholesterol content of the solution or serum (20 µL) was determined using the Cholesterol E-test Wako (optical density (OD) 600 nm).

Isolation of HDL and VLDL/LDL Fractions from Serum

The HDL and very low-density lipoprotein (VLDL)/LDL fractions were isolated from serum with the HDL and VLDL/LDL Quantification Kit, and their cholesterol content measured (OD 570 nm).

Statistical Analysis

Statistical analyses were performed using the Student’s t-test (Microsoft Excel; Microsoft Japan Co., Ltd.). Differences were considered significant at p<0.05. Data are presented as the mean±standard deviation (S.D.).

RESULTS

Comparison of Cholesterol Synthesis from [¹⁴C]Isopentenyl Pyrophosphate in WKY and SHRSP Livers

We initially investigated whether the reduced synthesis of cholesterol from isopentenyl pyrophosphate occurred in the liver of SHRSP. As shown in Fig. 2, less [¹⁴C]isopentenyl pyrophosphate was incorporated into cholesterol in the liver of SHRSP than in the liver of WKY. Our results indicated a reduction in enzyme levels downstream of MPD. Also, this result corresponded to the results of the reduction of mRNA levels of cholesterol biosynthetic enzyme as described by report.²⁵⁾

Fig. 2. Synthesis of Cholesterol from [¹⁴C]Isopentenyl Pyrophosphate in WKY and SHRSP Livers

Supernatants (10 mg) prepared from liver crude extracts were incubated with 1 mM [¹⁴C]isopentenyl pyrophosphate (1 µCi) aerobically, and the radioactivities incorporated into total cholesterol were measured. The values shown are the averages of duplicates that varied by less than 10%. ●: WKY, ■: SHRSP.

Comparison of the mRNA Levels of Cholesterol Biosynthetic Enzymes in the Liver of WKY and SHRSP

To examine whether the reduced cholesterol biosynthetic enzymes downstream of MPD in the livers of SHRSP occurred as previously reported,²⁵⁾ we measured the mRNA levels in the liver of SHRSP and WKY by the real-time PCR using primers in Table 1. Although the mRNA levels of all the cholesterol biosynthetic enzymes in SHRSP were lower than those in WKY, FPS and SQE were significantly lower in SHRSP than in WKY (Fig. 3). There was no marked difference of IPI, SQS, LSS, sterol C5 desaturase (SC5D) and 7-dehydrocholesterol reductase (7DHCR). No significant differences were observed in the mRNA levels of sterol regulatory element binding protein-2 (SREBP-2; mainly involved in cholesterol synthesis), which is a transcriptional factor of cholesterol biosynthesis enzymes containing HMG-CoA reductase, between SHRSP and WKY (Fig. 3). The results of mRNA levels of IPI, SQS, LSS and SREBP-2 differed from the data of Nemoto et al.²⁵⁾

Fig. 3. mRNA Levels of Cholesterol Biosynthetic Enzymes in the Liver of WKY and SHRSP

Real time-PCR was performed with the primer pairs listed in Table 1 using total RNA from the livers of SHRSP and WKY, as described in Materials and Methods. Each PCR resulted in a single band. Relative gene expression was quantified using GAPDH as an internal control. Values are the mean±S.D. of four independent experiments. Significant differences: * p<0.05.

Comparison of the Protein Levels of FPS and SQE in the Liver of WKY and SHRSP

We investigated whether the decrease in mRNA levels is accompanied with a reduction in protein levels. SQE protein levels in the liver of SHRSP were significantly lower than those in WKY, whereas FPS protein levels in the liver were not (Fig. 4). From these data, it was suggested that the reduced SQE protein levels in the liver of SHRSP were due to a decrease in mRNA levels. Reductions in the protein levels of SQE may also have participated in reducing cholesterol biosynthesis in the livers of SHRSP.

Fig. 4. Protein Levels of FPS and SQE in the Liver of WKY and SHRSP

Samples (A: FPS and SQE; 50 µg, GAPDH; 5 µg) were subjected to immunoblotting using the anti-FPS, anti-SQE, or anti-GAPDH antibody and signals were measured using an Intelligent Quantifier (B). Relative protein levels were quantified using GAPDH as an internal control. Values are the mean±S.D. of four independent experiments. Significant differences: * p<0.05.

Comparison of Nucleotide Sequences of Exons in SQE Genes of WKY and SHRSP

Changes in the amino acid sequence of SQE have been suggested to decrease its activity. Thus, we investigated whether a mutation existed in the nucleotide sequence of the exon in the SQE genes of WKY and SHRSP using the complete genome sequence data of WKY and SHRSP as described previously (purchased from the Society for Hypertension-related Disease Model research).³⁴⁾ As shown in Fig. 5, the mutation of one base was detected in exon 5 of the SQE gene of SHRSP, however, no changes in amino acids due to the mutation was observed between WKY (TCC; serine) and SHRSP (TCT; serine). No significant difference was observed in exons (exons 1–4, 6–11) other than exon 5 in the SQE gene between WKY and SHRSP. On the other hand, when PCR was performed using the SQE primer (F-CAC CAA AGA AGC TTT TGA GCC ATG TGG ACT/R-ACT TCC ATG TGG TTT CCC TTT CAG TGA AC) and cDNA was prepared from total RNA (mRNA) from the livers of WKY and SHRSP, the base pairs (bp) of PCR products (bp of the full length of SQE containing start and stop codons) in SHRSP were the same as those in WKY (data not shown). Furthermore, when PCR products were treated with various restriction endonucleases (exon 1; PvuII, exon 2; PstI, exons 3,11; NcoI, exon 5; SacI, exon 6; AflII, exons 7, 10; HindIII, exons 8, 9; DdeI), the bp of all fragments in SHRSP were similar to those in WKY (data not shown). These results suggested that reductions in SQE activity by the mutation of an amino acid and alternative splicing did not occur in SHRSP. Namely, reductions in the activity of SQE in the livers of SHRSP may have been due to lower amounts of the SQE protein.

Fig. 5. Comparison of Nucleotide Sequences of Exon 5 in SQE Genes of WKY and SHRSP

A: The nucleotide sequence of exon 5 (114 bp) in the SQE gene in WKY. Underlined is the codon-containing base sequence of the mutation between WKY and SHRSP. B: The codon containing the base sequence of the mutation between WKY (TCC) and SHRSP (TCT). Underlined is the base sequence of the mutation between WKY (C) and SHRSP (T). The codon-containing base sequence of the mutation did not have a different amino acid sequence between WKY (TCC; serine) and SHRSP (TCT; serine).

Comparison of 2,3-Oxidosqualene Synthesis from [¹⁴C]Isopentenyl Pyrophosphate in WKY and SHRSP Livers

If reductions in SQE activity were due to decreases in SQE protein levels, the synthesis of 2,3-oxidosqualene from isopentenyl pyrophosphate may also be decreased in the livers of SHRSP. As shown in Fig. 6, the synthesis of 2,3-oxidosqualene in the liver was significantly lower in SHRSP than in WKY. As described above, no significant differences were observed in the mRNA levels of IPP and SQS in the liver between SHRSP and WKY (Fig. 3). Furthermore, FPS protein levels in the liver were similar in SHRSP and WKY (Fig. 4). These results strongly suggested that the lower amounts of 2,3-oxidosqualene in the livers of SHRSP were due to decreases in SQE activity, which were attributed to lower protein levels of SQE.

Fig. 6. Synthesis of 2,3-Oxidosqualene from [¹⁴C]Isopentenyl Pyrophosphate in WKY and SHRSP Livers

Supernatants (10 mg) prepared from liver crude extracts were incubated with 1 mM [¹⁴C]isopentenyl pyrophosphate (0.5 µCi), and the radioactivities incorporated into 2,3-oxidosqualene were measured. Values are the mean±S.D. of four independent experiments. Significant differences: * p<0.05.

Comparison of LDLR, SRB1, and Abca1 mRNA and Protein Levels in WKY and SHRSP Livers

We previously reported that total cholesterol (40%), HDL-cholesterol (40%), and VLDL/LDL-cholesterol (20%) levels in the serum and cholesterol content (20%) in the liver were significantly lower in SHRSP than in WKY.²²⁾ Similar results (results of serum total cholesterol, HDL-, or VLDL/LDL-cholesterol) were obtained between WKY and SHRSP in the present study (data not shown). In order to identify the causes of these reductions in serum cholesterol and cholesterol content in the livers of SHRSP, the mRNA levels of LDLR, SRB1, and Abca1 were measured in the livers of SHRSP and WKY using real-time PCR with primers, as shown in Table 1. The mRNA levels of SRB1 in the liver were significantly higher in SHRSP than in WKY, whereas no marked changes were noted in LDLR and Abca1 (Fig. 7). Furthermore, when LDLR, SRB1, and Abca1 protein levels were measured in SHRSP and WKY livers by an immunoblot analysis, SRB1 protein levels in SHRSP livers were also significantly increased; however, no remarkable differences were observed in LDLR and Abca1 protein levels as well as mRNA levels (Fig. 8). These results suggested that increases in the uptake of HDL-cholesterol from the serum to the liver due to increases in SRB1 protein levels in SHRSP livers may play a role in reducing serum cholesterol levels (decreases in total cholesterol and HDL-cholesterol).

Fig. 7. LDLR, SRB1, and Abca1 mRNA Levels in WKY and SHRSP Livers

Real time-PCR was performed with the primer pairs listed in Table 1 using total RNA from the livers of SHRSP and WKY, as described in Materials and Methods. Each PCR resulted in a single band. Relative gene expression was quantified using GAPDH as an internal control. Values are the mean±S.D. of four independent experiments. Significant differences: * p<0.05.

Fig. 8. LDLR, SRB1, and Abca1 Protein Levels in WKY and SHRSP Livers

Samples (A: LDLR, SRB1, and Abca1; 50 µg, GAPDH; 5 µg) were subjected to immunoblotting using the anti-LDLR, anti-SRB1, anti-Abca1, or anti-GAPDH antibody, and signals were measured using an Intelligent Quantifier (B). Relative protein levels were quantified using GAPDH as an internal control. Values are the mean±S.D. of four independent experiments. Significant differences: * p<0.05.

Comparison of the mRNA Levels of Cholesterol Biosynthetic Enzymes in the Testis of WKY and SHRSP

We previously reported that MPD levels were significantly lower in the brains as well as liver of SHRSP, when the tissue distribution of MPD was examined in WKY and SHRSP.²⁶⁾ In order to determine whether reductions in SQE or cholesterol biosynthetic enzymes downstream of MPD occurred in tissues other than the liver, we examined the mRNA levels of cholesterol biosynthetic enzymes in the testis of SHRSP and WKY using real-time PCR. SQE mRNA levels in the testis were significantly lower in SHRSP than in WKY (Fig. 9). These results indicated that reductions in the mRNA levels of cholesterol biosynthetic enzymes differed in each tissue.

Fig. 9. mRNA Levels of Cholesterol Biosynthetic Enzymes in the Testis of WKY and SHRSP

Real time-PCR was performed with the primer pairs listed in Table 1 using total RNA from the testis of SHRSP and WKY, as described in Materials and Methods. Each PCR resulted in a single band. Relative gene expression was quantified using GAPDH as an internal control. Values are the mean±S.D. of four independent experiments. Significant differences: * p<0.05.

Comparison of the Protein Levels of SQE in Tissues of WKY and SHRSP

In order to establish whether the lower protein levels of SQE occurred in tissues of SHRSP, we measured the amount of SQE in tissues (brain, lung, heart, pancreas, spleen, kidney, and testis) by immunoblot analysis between WKY and SHRSP. The protein levels of SQE in tissues other than the brain were lower in SHRSP than in WKY (Fig. 10). These results suggested that the decrease in cholesterol biosynthesis in tissues other than the brains of SHRSP was caused by reductions in the protein levels of SQE.

Fig. 10. Immunoblot Analysis of SQE Protein Levels in WKY and SHRSP Tissues

Tissue samples (A: SQE; 50 µg, GAPDH; 5 µg [brain], 2 µg [lung], 4 µg [heart], 3.5 µg [pancreas], 2.5 µg [spleen], 2 µg [kidney], and 3.5 µg [testis]) in WKY (W) and SHRSP (SP) were subjected to immunoblotting using an anti-SQE or anti-GAPDH antibody, and signals were measured using an Intelligent Quantifier (B). Relative protein levels were quantified using GAPDH as an internal control. Values are the mean±S.D. of four independent experiments. Significant differences: * p<0.05.

Comparison of Cholesterol Contents, the Protein Levels of LDLR and Abca1 in Testis of WKY and SHRSP

To investigate whether the decrease of cholesterol contents in tissues of SHRSP was caused by the reduction of protein levels of SQE, we determined the cholesterol contents in the testis between WKY and SHRSP. Cholesterol contents in the testis of SHRSP were the same as those of WKY (Fig. 11). These results suggested that cholesterol contents in the testis of SHRSP are maintained/supplemented by the accelerated uptake of cholesterol in the serum and the suppressed efflux of cholesterol from the testis to the serum. Thus, we measured protein levels of LDLR (involved in uptake) and Abca1 (participate in efflux) in the testis of SHRSP and WKY using an immunoblot analysis. No significant differences were observed in LDLR or Abca1 protein levels in the testis between SHRSP and WKY (Fig. 12). These results suggested that LDLR and Abca1 were not involved in maintaining cholesterol contents in the testis of SHRSP.

Fig. 11. Cholesterol Contents in the Testis of WKY and SHRSP

Cholesterol contents in the testis of WKY and SHRSP measured by Cholesterol E-test Wako as described in Materials and Methods. Values are the mean±S.D. of four independent experiments.

Fig. 12. LDLR and Abca1 Protein Levels in the Testis of WKY and SHRSP

Samples (A: LDLR and Abca1; 50 µg, GAPDH; 5 µg) were subjected to immunoblotting using an anti-LDLR, anti-Abca1, or anti-GAPDH antibody, and signals were measured using an Intelligent Quantifier (B). Relative protein levels were quantified using GAPDH as an internal control. Values are the mean±S.D. of four independent experiments.

DISCUSSION

The protein levels of FPS in the livers of SHRSP did not correlate with the decrease in FPS mRNA levels (Figs. 3, 4). No significant difference was noted in FPS protein levels in SHRSP livers (Fig. 4). Therefore, we considered that degradation of the FPS proteins was less in SHRSP (inhibiting the degradation of FPS protein levels) than in WKY. Lastres-Becker et al. reported that the reduction of insulin receptor expression in ataxin-2 deficient mouse caused by a post-transcriptional effect as protein level was decreased, although the mRNA levels was increased.³⁵⁾

Nemoto et al. reported that mRNA levels of IPI, FPS, SQS, SQE and LSS in the liver of SHRSP were significantly lower than (less than 50%) those in WKY.²⁵⁾ However, our results did not significantly show the reduction of mRNA levels of IPI, SQS and LSS (Fig. 3). We previously reported that MPD protein levels in the livers of SHRSP were reduced from as early as 2 weeks of age, but not a week of age.²⁶⁾ Therefore, the reduction in the mRNA levels of cholesterol biosynthetic enzymes in the liver of SHRSP may differ by week old. Also, the difference of results of the mRNA level may occur by experimental conditions (week old of rats, primers using real-time PCR or storage condition and storage time of sample). Although the lower mRNA levels of SREBP-2 were reported in SHRSP, there was no difference in this study (Fig. 3). From our results, SREBP-2 in the liver of SHRSP was not considered important as a cause of reducing mRNA levels of cholesterol biosynthetic enzymes.

SQE protein and mRNA levels in the liver were significantly lower in SHRSP than in WKY (Figs. 3, 4). We suggested that reductions in mRNA levels were an important cause of the decrease in SQE protein levels in the livers of SHRSP. We hypothesized that lower SQE mRNA levels in the livers of SHRSP were caused by changes/mutations in the silencer element or enhancer element in the promoter region of SQE if the reduction in SQE mRNA levels that occurred due to a change in the promoter is considered. Lower SQE mRNA levels in the livers of SHRSP may also have been caused by an increase in repressor binding to the silencer element in the promoter sequence of SQE or a decrease in activator binding to the enhancer element in the promoter region of SQE if the reduction in SQE mRNA levels that occurred due to the change in transcriptional factors is considered, but not the promoter region. Furthermore, lower SQE protein levels in the livers of SHRSP may have occurred due to accelerated protein degradation or another regulation mechanism. microRNA (miR) was recently shown to reduce protein levels by inhibiting its synthesis from mRNA or accelerating the degradation of mRNA.^23,24) A large number of miR were previously detected when miR combined with the 3′-untranslated region of SQE was searched for using microRNA.org.²²⁾ Twelve miR (21, 124, 133a, 133b, 200a, 200b, 200c, 205, 224, 377, 429, and 494) combined with the 3′-untranslated region of SQE. The lower SQE mRNA levels in the livers of SHRSP may have been caused by an increase in miR combined with the 3′-untranslated region of SQE if the reduction in SQE mRNA levels plays a role in miR. The upregulation of SRB1 may also have been caused by a change in the protein region, transcriptional factor, or miR. A recent study reported that the overexpression of miR (96, 125a, 185, 223, 455) reduced protein levels of SRB1 and the uptake of HDL-cholesterol.^36,37) Therefore, higher SRB1 mRNA levels in SHRSP livers may have been caused by a decrease in miR combined with the 3′-untranslated region of SRB1 if the increase in SRB1 mRNA levels does indeed play a role in miR.

In the present study, we indicated that the decrease of cholesterol contents was not caused in the testis of SHRSP (Fig. 11), although the reduction of protein levels of SQE occurred in testis of SHRSP (Fig. 10). No marked differences were observed in LDLR and Abca1 levels in the testis of SHRSP (Fig. 12). These results suggested that decreases in cholesterol contents due to lower SQE protein levels in the testis of SHRSP were supplemented by a mechanism other than SQE, LDLR, and Abca1. If the accelerated uptake of cholesterol from the serum to tissues (other than by LDLR) or the suppressed efflux of cholesterol from tissues to the serum (other than by Abca1) occurs to supplement decreases in cholesterol contents due to reductions in SQE protein levels in tissues other than the brain of SHRSP, reductions in SQE protein levels in tissues is an important factor causing lower levels of serum cholesterol. We previously compared the tissue distribution of MPD between WKY and SHRSP and showed that MPD protein levels were lower in the brains and livers of SHRSP.²⁶⁾ Since the amount of SQE was low in tissues other than the brain, a lower amount of SQE rather than that of MPD may be an important main cause of reductions in serum cholesterol, whereas reductions in MPD in the liver played a role in decreasing serum cholesterol levels. Further studies are needed in order to elucidate the mechanisms maintaining/supplementing cholesterol contents in other tissues (SQE-deficient tissues) such as the testis.

In the present study, we showed that lower SQE and higher SRB1 protein levels in SHRSP livers may mainly be responsible for the reduction in serum cholesterol levels in these animals. Thus, we considered reasons for/causes of the reductions in serum cholesterol levels in SHRSP. The upregulation of SRB1 protein levels may occur in order to supplement decreases in cholesterol contents due to reductions in SQE and MPD protein levels in SHRSP livers. This may also have resulted in reductions in serum HDL-cholesterol in SHRSP. However, cholesterol contents in the liver were lower in SHRSP than in WKY, in spite of increases in the uptake of HDL-cholesterol from the serum to the liver due to the upregulation of SRB1. Therefore, the release of VLDL/LDL-cholesterol from the liver to the serum may have been decreased, leading to reductions in cholesterol contents in SHRSP livers. Reductions in total cholesterol containing HDL (mainly) and LDL were caused by these mechanisms (reductions in SQE and MPD and increases in SRB1 in SHRSP livers). Further studies are needed in order to elucidate the mechanisms responsible for reducing serum cholesterol and SQE expression levels, or increasing SRB1 expression levels in SHRSP livers.

Acknowledgment

This work was supported by Fukuyama University Grant for Academic Research Projects (GARP).

Conflict of Interest

The authors declare no conflict of interest.

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