Involvement of Hepatic IL-1 in the Strain-Dependent Sex Differences in Serum Total Cholesterol Levels in Rats

Abstract

The strain and sex differences in serum total cholesterol (TC) levels were examined in F344 and Sprague-Dawley (SD) rats. A sex difference (male<female) was observed in F344 rats but not in SD rats. The strain-dependent sex difference (male<female) was also observed in the constitutive gene expression level of hepatic 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR), a rate-limiting enzyme for cholesterol biosynthesis, but not in the expression of hepatic cholesterol 7α-hydroxylase, a rate-limiting enzyme for cholesterol catabolism (bile acid biosynthesis from cholesterol). The strain-dependent sex difference in hepatic HMGR gene expression was closely correlated with the levels of hepatic interleukins (ILs), especially of IL-1α, which acts as a positive regulator for the hepatic HMGR gene. Hepatic IL-1α protein expression was higher in female F344 rats than in male F344 rats and compared with male and female SD rats. Similar to hepatic IL-1α protein expression, serum TC levels were highest in female F344 rats than in the other groups of rats. Serum TC and hepatic IL-1α levels in male F344 rats were similar to those in male and female SD rats. The present findings demonstrate for the first time that strain-dependent sex difference in serum TC level between F344 and SD rats is, at least in part, related to difference in the IL-1α-mediated HMGR gene expression level in the liver.

Cholesterol is not only an essential component of biological membranes but also a precursor for all steroid hormones. Serum cholesterol homeostasis is tightly controlled by the coordinated regulation of biosynthesis, uptake, and metabolism of cholesterol.^1,2) The gene expression of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR), a rate-limiting enzyme involved in cholesterol biosynthesis, is controlled through feedback regulation by oxysterols, including cholesterol.¹⁾ The gene expression of cholesterol 7α-hydroxylase (CYP7A1), a rate-limiting enzyme for bile acid biosynthesis from cholesterol, is controlled by feedforward regulation by oxysterols and feedback regulation by bile acids.^2,3) Despite these overall patterns of regulation, the serum total cholesterol (TC) level in animals, including humans, shows marked sex differences. In most mice strains and in humans, the serum TC level is much higher in the male sex than in the female sex,^4–7) whereas the opposite is found in rats and pigs.^8,9) To date, however, the causes of these sex and species differences remain unclear.

It was reported that inflammatory cytokines, including interleukin-1 (IL-1), alter the gene expression levels of HMGR and CYP7A1.^10,11) More recently, Kojima et al.⁷⁾ demonstrated that the constitutive serum TC levels were higher in IL-1α/β-knockout (IL-1-KO) BALB/c mice than in sex-matched wild-type mice, and that hepatic IL-1 levels were significantly higher in female than in male wild-type BALB/c mice, while opposite results were found for the sex difference in serum TC levels. Taken together, the results of these previous studies suggest that inflammatory cytokines, especially IL-1, play an important role in controlling the constitutive serum TC level by regulating the gene expression levels of hepatic enzymes, especially HMGR and CYP7A1 that are responsible for cholesterol biosynthesis and catabolism.

In this study, we focused on hepatic inflammatory cytokines, especially IL-1, and examined the relationships among cytokine levels, serum TC levels, and hepatic HMGR and CYP7A1 gene expression levels in male and female F344 and Sprague-Dawley (SD) rats. F344 rats, but not SD rats, are known to show a sex difference (male<female) in serum TC levels.^8,12,13) The present findings is the first to demonstrate a strain-dependent sex difference in serum TC levels between F344 and SD rats, a difference that is at least partly related to differences in hepatic IL-1α-mediated HMGR gene expression.

MATERIALS AND METHODS

Animals

F344 rats were purchased from Japan SLC Inc. (Hamamatsu, Japan), and SD rats were from Charles River Japan (Atsugi, Japan). The rats were obtained at 11 weeks of age and were used at 12 weeks of age. Rats were kept in an air-conditioned room with a 12-h light/dark cycle. They were fed a commercial chow (CRF-1; Oriental Yeast Co., Ltd., Tokyo, Japan) and water ad libitum. All animals were handled humanely in accordance with the animal care guidelines of Kaken Pharmaceutical Co., Ltd. (Shizuoka, Japan).

Serum TC

Rats were killed between 10 : 00 a.m. and 12 : 00 a.m. by exsanguination of the carotid artery under anesthesia with carbon dioxide. Blood samples from individual rats were collected in a Separapid tube (Sekisui Chemical Co., Osaka, Japan), and their sera were prepared by centrifugation at 2000×g for 10 min. The sera obtained were stored at −80°C until use. The serum TC level was measured with a 7170 Automatic Analyzer (Hitachi, Tokyo, Japan) using L-Type CHO H reagent (Wako Pure Chemical Industries, Ltd., Osaka, Japan).

Real-Time Reverse-Transcription-Polymerase Chain Reaction (RT-PCR) Analyses of Hepatic HMGR, CYP7A1, and IL-1 mRNAs

Rats were killed between 10 : 00 a.m. and 12 : 00 a.m. as described above. The liver was rapidly removed from each rat, frozen in liquid nitrogen, and stored at −80°C until use. Total RNA was prepared from the liver using an RNAeasy® minikit (Qiagen, Tokyo, Japan) according to the manufacturer’s instructions. Total RNA was converted to cDNA with a QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer’s instructions, and the resultant cDNAs were used for real-time RT-PCR. Real-time RT-PCR was performed in reaction mixture (total volume 25 µL) containing 10 ng cDNA and 0.5 µM of each primer set (Rn_Hmgcr_1_SG for HMGR, Rn_Cyp7a1_1_SG for CYP7A1, Rn_Il1a_1_SG for IL-1α, Rn_Il1b_1_SG for IL-1β, and Rn_Actb_1_SG for β-actin; QuantiTect primer assay, Qiagen) with a Mini Opticon™ Real-Time PCR Detection System (Bio-Rad Laboratories GmbH, Munich, Germany) using a QuantiFast™ SYBR® Green PCR Kit (Qiagen). The amount of each cDNA was assessed by the relative-standard curve method using β-actin as an internal standard.

Hepatic IL-1 Proteins

The protein expression levels of hepatic IL-1α and IL-1β were measured using Quantikine® Rat IL-1α and IL-1β immunoassay kits (R&D Systems, Minneapolis, MN, U.S.A.), as previously described.¹⁴⁾ Briefly, the liver from each rat was homogenized in two volumes (w/v) of 1.15% KCl. The liver homogenate was centrifuged at 9000×g for 20 min at 4°C, and the supernatant was centrifuged at 105000×g for 1 h at 4°C. The resulting supernatant (termed S-105) was used to measure IL-1α and IL-1β levels using the immunoassay kits. The total protein level was also measured using a Pierce® BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, U.S.A.).

Serum Sex Hormones

The serum estradiol and testosterone levels were measured using estradiol and testosterone enzyme immunoassay (EIA) kits (Cayman Chemical Company, Ann Arbor, MI, U.S.A.). To measure estradiol levels, serum was extracted twice with methylene chloride, after which the methylene chloride was evaporated. The residue was dissolved in EIA buffer for the EIA. To measure testosterone levels, serum was diluted several times with EIA buffer for the EIA.

Statistical Analysis

Significant differences between two experimental groups were evaluated using the Student’s t-test. Correlations between the indicated parameters were determined by regression analysis. All statistical analyses were done using GraphPad Prism 4 software (GraphPad Software, San Diego, CA, U.S.A.).

RESULTS

Serum TC Levels

The serum TC levels in both sexes of F344 and SD rats are shown in Fig. 1. In F344 rats, the serum TC level was significantly higher in female rats (98±3 mg/dL) than in male rats (80±2 mg/dL). By contrast, there was no difference in the serum TC between male and female SD rats (about 73 mg/dL in both sexes).

Fig. 1. Serum TC Levels in Male and Female F344 and SD Rats

The TC level was measured in blood samples obtained from 12 rats per sex and strain. Closed and open boxes indicate the mean values for male (M) and female (F) rats, respectively. Bars represent standard deviations. Asterisks indicate significant differences between male and female rats (* p<0.0001).

Gene Expression Levels of Hepatic HMGR and CYP7A1

We first examined the sex differences in hepatic HMGR and CYP7A1 gene expression levels, which encode the rate-limiting enzymes for cholesterol biosynthesis and bile acid biosynthesis from cholesterol, respectively, in F344 rats. The hepatic HMGR gene expression level was significantly higher, by about 1.6 times, in female than in male rats, whereas no sex difference was observed for CYP7A1 (Fig. 2A). There were no sex differences in the expression levels of any of the genes examined in SD rats (Fig. 2B).

Fig. 2. Hepatic HMGR and CYP7A1 mRNA Expression Levels in F344 (A) and SD Rats (B)

Total RNA was isolated from liver samples collected from 12 rats per sex and strain and was used for real-time RT-PCR analysis. The mRNA expression levels of HMGR and CYP7A1 are shown as the ratios to levels of β-actin, which was used as the internal standard. Closed and open boxes indicate the mean values for male (M) and female (F) rats, respectively. Bars represent standard deviations. Asterisks indicate significant differences between male and female rats (* p<0.0001).

Hepatic IL-1α/β Levels

Hepatic IL-1α and IL-1β gene expression levels were assessed in both sexes of F344 and SD rats. In F344 rats, hepatic IL-1α and IL-1β gene expression levels were higher in females than in males (Fig. 3A), whereas no sex differences were observed in SD rats (Fig. 3B). Consistent with the gene expression levels, hepatic IL-1α protein levels were significantly higher in female than in male F344 rats (Fig. 3C), while no sex difference was observed for IL-1β. In SD rats, there was no sex difference in the IL-1α protein level, but the IL-1β protein level was higher in females than in males (Fig. 3D).

Fig. 3. Hepatic mRNA and Protein Expression Levels of IL-1α and IL-1β in F344 and SD Rats

Total RNA and S-105 was prepared from liver samples obtained from 12 rats per sex and strain. Total RNA and S-105 were used for real-time RT-PCR analysis and EIAs, respectively. The mRNA expression levels of IL-1α and IL-1β are shown as the ratios to the levels of β-actin, which was used as an internal standard. IL-1α/β proteins are shown as pg protein per mg S-105 protein. Closed and open boxes indicate the mean values for male (M) and female (F) rats, respectively. Bars represent standard deviations. Asterisks indicate significant differences between male and female rats (* p<0.01, ** p<0.001, and *** p<0.0001).

Relationships among the Hepatic IL-1 Protein Levels, Serum TC Levels, and Hepatic HMGR Gene Expression

In F344 rats, the hepatic IL-1α protein level was correlated with the serum TC level (Fig. 4A) and HMGR gene expression (Fig. 4B). Such correlations were not observed for the hepatic IL-1β protein level (Figs. 4C, D). A clear positive correlation between the hepatic HMGR gene expression level and the serum TC level was observed in F344 rats (Fig. 4E). By contrast, no such correlations were observed in SD rats (data not shown).

Fig. 4. Correlations among Hepatic IL-1α/β Protein Levels, Serum TC Levels, and Hepatic HMGR mRNA Expression Levels in F344 Rats

The correlations between hepatic IL-1α/β protein levels and either serum TC levels (A and C) or hepatic HMGR gene expression levels (B and D), and between hepatic HMGR gene expression levels and serum TC levels (E) were examined using the data shown in Figs. 1–3. Correlation coefficients were determined by regression analysis. r, correlation coefficient. Closed and open circles represent male and female rats, respectively.

Serum Sex Hormone Levels

The serum testosterone and estradiol levels in both sexes of F344 and SD rats were compared. In both strains of rats, the serum testosterone level was significantly higher in males than in females (Fig. 5A). The testosterone level was significantly higher in male SD rats than in male F344 rats, although the levels were similar between the female rats of both strains. The serum estradiol level was not significantly different between sex-matched F344 and SD rats, although it was higher in females than in males in each strain of rats (Fig. 5B).

Fig. 5. Serum Sex Hormone Levels in F344 and SD Rats

Serum testosterone and estradiol levels were measured in serum samples obtained from 12 rats per sex and strain. Closed and open boxes indicate the mean values for male (M) and female (F) rats, respectively. Bars represent standard deviations. Asterisks indicate significant differences between male and female rats (* p<0.05, ** p<0.01, and *** p<0.0001).

DISCUSSION

In this study, we first confirmed that there is a sex difference (male<female) in serum TC levels in F344 rats.^8,12) In general, the constitutive serum TC level is tightly maintained through a coordinated balance between the biosynthesis and catabolism of cholesterol in the liver.^1–3) Therefore, we focused on hepatic HMGR and CYP7A1, which are the rate-limiting enzymes for cholesterol biosynthesis and catabolism, respectively, and examined whether there are sex differences in the expression levels of the genes encoding these enzymes. We found a significant sex difference (male<female) in the hepatic gene expression level of HMGR but not of CYP7A1. Therefore, the sex difference (male<female) in the gene expression level of HMGR, but not of CYP7A1, in the F344 rat liver seems to contribute to the sex difference in the serum TC level. By contrast, in BALB/c mice, the sex difference (male>female) in the serum TC level was at least partly dependent on the difference in the hepatic gene expression of CYP7A1 (male<female) but not of HMGR.⁷⁾ Unfortunately, there is still no clear explanation for these species differences, although species differences in the structure, activity, and level of sex-associated regulatory factor(s) might influence the hepatic gene expression levels of CYP7A1 and/or HMGR.

An earlier study using IL-1-KO mice demonstrated that hepatic proinflammatory cytokines, especially IL-1, play crucial roles in the constitutive regulation of serum TC levels and of hepatic HMGR and CYP7A1 gene expression levels in mice.⁷⁾ Administration of IL-1 or an IL-1 inducer was reported to increase hepatic HMGR gene expression and serum TC levels in hamsters,¹⁰⁾ rats,¹⁴⁾ and mice.¹⁵⁾ Therefore, we investigated whether there are sex differences in the hepatic mRNA and protein levels of IL-1α and IL-1β in F344 rats. In these analyses, we found marked sex differences (male<female) in the hepatic mRNA and protein levels of IL-1α. In addition, the IL-1α protein level was positively correlated with the serum TC level. Regarding hepatic IL-1β, we found a sex difference for its mRNA level but not for its protein level. Incidentally, it has been reported that the expression level of IL-1β protein is not necessarily correlated with that of its mRNA.¹⁶⁾ Moreover, in this study, no significant correlation between hepatic IL-1β protein level and serum TC level was observed. These present findings strongly suggest that the sex difference in the hepatic IL-1α level may contribute to the sex difference in the serum TC level in F344 rats.

This hypothesis was further supported by the present findings showing the absence of sex differences in hepatic IL-1α protein and serum TC levels in SD rats. Strain-specific sex differences in serum TC levels were also reported in other rats.^8,12,13) In this study, we also found that the hepatic IL-1α protein level was significantly higher in female F344 rats than in male F344 rats and compared with male and female SD rats. These findings also support the hypothesis proposed above.

Despite these findings, the exact cause(s) of the strain-dependent sex differences between F344 and SD rats in hepatic IL-1α mRNA and protein levels remain unclear. Although the serum androgen levels were significantly higher in male SD rats than in male F344 rats, there were no sex differences in hepatic IL-1α mRNA and protein levels in SD rats. Furthermore, there were no significant differences in serum estrogen levels between sex-matched F344 and SD rats. These findings suggest that the strain-dependent sex differences in hepatic IL-1α mRNA and protein levels are not solely mediated by differences in sex hormone levels. However, it was reported that IL-1 secretion from peritoneal macrophages obtained from Lewis rats was higher in female rats than in male rats.¹⁷⁾ It was also reported that IL-1 expression was increased by estradiol in human rheumatoid fibroblast-like synovial cells and human breast tissues^18,19) and the estradiol-induced increase of the uterus weight was greater in ovariectomized F344 rats than in ovariectomized SD rats.²⁰⁾ In addition, it was suggested that the strain difference in the responses to estrogen might involve the difference in the expression of arginine methyltransferase 1, which acts as a co-activator of estrogen receptor (ER)α and ERβ.²¹⁾ Therefore, it is important to elucidate possible differences in receptor activities and/or their associated sex hormone levels between F344 and SD rats. Studies examining the polymorphisms of sex hormone receptor genes and/or related factors are also needed.

In conclusion, we have demonstrated that F344 rats, but not SD rats, exhibit sex differences (male<female) in serum TC, hepatic HMGR gene expression, and hepatic IL-1α protein levels. Additionally, these parameters were positive correlated with each other in F344 rats, but not in SD rats. Therefore, our results indicate that hepatic IL-1α plays an important role in the sex-dependent regulation of hepatic HMGR gene and serum TC levels in F344 rats.

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