0013-7227/04/$15.00/0 Endocrinology 145(4):1972–1979 Printed in U.S.A. Copyright © 2004 by The Endocrine Society doi: 10.1210/en.2003-0874 Female-Predominant Expression of Fatty Acid Translocase/CD36 in Rat and Human Liver NINA STÅHLBERG, ELIZABETH RICO-BAUTISTA, RACHEL M. FISHER, XUXIA WU, LOUISA CHEUNG, AMILCAR FLORES-MORALES, GUNNEL TYBRING, GUNNAR NORSTEDT, AND PETRA TOLLET-EGNELL Department of Molecular Medicine (N.S., E.R.-B., X.W., L.C., A.F.-M., G.N., P.T.-E.), and Atherosclerosis Research Unit, Downloaded from https://academic.oup.com/endo/article/145/4/1972/2878908 by guest on 01 October 2021 King Gustaf V Research Institute (R.M.F.), Karolinska Hospital, Karolinska Institute, SE-171 76 Stockholm, Sweden; and Department of Laboratory Medicine, Division of Clinical Pharmacology, Huddinge University Hospital, Karolinska Institute (G.T.), SE-141 86 Stockholm, Sweden The aim of this study was to identify genes for hepatic fuel predominant transcripts was fatty acid translocase/CD36, metabolism with a gender-differentiated expression and to with 18-fold higher mRNA levels in the female liver and 4-fold determine which of these that might be regulated by the fe- higher mRNA levels in males treated with GH, compared with male-specific secretion of GH. Effects of gender and continu- untreated males. This gender-differentiated expression was ous infusion of GH to male rats were studied in the liver using confirmed at mRNA and protein levels in the rat and at the cDNA microarrays representing 3200 genes. Sixty-nine tran- mRNA level in human livers. Although purely speculative, it scripts displayed higher expression levels in females, and 177 is possible that higher levels of fatty acid translocase/CD36 in displayed higher expression in males. The portion of GH- human female liver might contribute to the sexually dimor- regulated genes was the same (30%) within the two groups of phic development of diseases resulting from or characterized gender-specific genes. The male liver had a higher expression by disturbances in lipid metabolism, such as arteriosclerosis, of genes involved in fuel metabolism, indicating that male rats hyperlipidemia, and insulin resistance. (Endocrinology 145: might have a greater capacity for high metabolic turnover, 1972–1979, 2004) compared with females. Most notable among the female- T IS WELL known that the body composition differs be- pattern) infusion of GH (8). Interestingly, these different I tween the sexes, and there is also increasing evidence that types of GH treatment induced opposite changes in the same fuel metabolism differs between genders (1–3). It is therefore set of genes. This implies that previously reported gender- likely that tissues from male and female individuals have related effects of GH, such as body growth, might include a different metabolic functions and that genes from different sexually dimorphic metabolism of lipids. metabolic pathways are expressed in a sex-specific manner. Gender differences are also seen in the manifestations of Hepatic sex differences at the level of gene expression have different disorders, some of which may have links to sex- previously been demonstrated and shown to be regulated by specific liver functions. The male predominance of the met- different hormones (4), including sex steroids and peptide abolic syndrome is well established, and men have an earlier hormones. One hormone in the latter category is GH. The onset and a higher incidence of coronary heart disease (9). existence of a sexually dimorphic pattern of GH secretion The sexually dimorphic development of lipid-related disor- was first described in the rat (5), but has also been demon- ders motivates investigations that seek to clarify gender dif- strated in other species, including humans (6). In adult male ferences in the physiology and pathophysiology of the liver. rats, GH is secreted in episodic bursts at 3- to 4-h intervals, Generating sex-specific transcript profiles should help to elu- with low or undetectable levels between peaks, whereas cidate molecular mechanisms behind sex-specific functions females have a more continuous pattern of secretion. In ro- of the liver. The aim of the present investigation was to dents, biological effects of this sexually differentiated pattern identify genes involved in the metabolism of carbohydrates, of GH secretion includes sex differences in body weight gain and longitudinal bone growth (7), but also hepatic steroid lipids, and protein with a gender-differentiated expression metabolism (4). We have recently demonstrated that episodic and to determine which of these might be regulated by the (male pattern) GH treatment of old male rats repress the sex-specific secretion of GH. We have collected transcript expression of hepatic gene products involved in lipogenesis profiles from male and female rat livers using cDNA mi- and induce genes for fatty acid oxidation (7a). In a related croarrays representing 3200 genes. Gender differences in study, old male rats were treated by continuous (female gene expression have been compared with the effect of con- tinuous infusion of GH to male rats. Most notable among the female-predominant gene products was fatty acid translo- Abbreviations: FAT/CD36, Fatty acid translocase/CD36; LCFA, long-chain fatty acid; RNase, ribonuclease. case/CD36 (FAT/CD36), which was shown to have 18-fold higher mRNA levels in the female liver. This gender-differ- Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the entiated expression was confirmed at mRNA and protein endocrine community. levels in the rat and at the mRNA level in humans. 1972 Ståhlberg et al. • Sex-Differentiated Expression in the Liver Endocrinology, April 2004, 145(4):1972–1979 1973 Materials and Methods to the method of Melton et al. (15). Samples were analyzed in triplicate, Animals and the results were determined as counts per minute of mRNA per microgram of total RNA. The results are expressed as the mean Ϯ sd, Ten-week-old male and female Sprague Dawley rats (B&K Universal and a t test was used for the determination of statistical significance. AB, Hull, UK) were maintained under standardized conditions. Five male rats were treated with bovine GH (gift from Pharmacia & Upjohn AB, Stockholm, Sweden) by continuous infusion from Immunoblotting osmotic minipumps (model 2001, B&K Universal AB) at a dose of 5 Tissue homogenates were resolved by SDS-PAGE on 7.5% polyacryl- ␮g/h. After 1 wk of treatment, the rats were killed, and tissues removed amide gels and transferred to polyvinylidene difluoride membranes and frozen in liquid nitrogen. The animal experiments were approved with a Trans-Blot SD semidry transfer cell (Bio-Rad Laboratories, Her- by the institutional animal care and use committee. cules, CA). The membranes were blocked for2hinTris-buffered saline Human liver samples (10 mm Tris, pH 8.0, and 150 mm NaCl) containing 0.05% (vol/vol) Tween 20 and 10% (wt/vol) milk powder, incubated for 2 h with mono- Human liver tissue was obtained from a donor liver bank established clonal anti-FAT/CD36 antibodies (antimurine CD36, Cascade Bio- at the Department of Clinical Pharmacology at Huddinge University sciences, Winchester, MA) diluted 1:2500 in Tris-buffered saline, Downloaded from https://academic.oup.com/endo/article/145/4/1972/2878908 by guest on 01 October 2021 Hospital. The liver specimens which for various reasons could not be washed, and incubated with the secondary antibody (1:10,000; donkey used for organ transplantation, included seven women and five men antimouse IgG, Jackson ImmunoResearch Laboratories, West Grove, between 22 and 50 yr of age. None of the subjects had been diagnosed PA). After additional washing steps antibody binding was visualized with metabolic disorders. The donors were checked for hepatitis B and using an ECL detection system (Pierce Chemical Co., Rockford, IL). C serology, and all were HIV negative. All livers were processed to be used for transplantation and were transfused with 5–10 liters Belzer UW Immunohistochemistry Cold Storage Solution (ViaSpan, DuPont Pharmaceuticals, Wilmington, DE). Before transfusion, benzyl penicillin (0.12 mg), actaprid (1.4 mg), Cryostat sections (5 ␮m) from rat livers were used in a standard and dexamethasone (16 mg) were added (all per liter of UW solution). immunohistochemical protocol (avidin-biotin-peroxidase). After rins- The livers were handled within the ischemic time of about 16 h, cut into ing the sections in PBS, nonspecific endogenous peroxidase activity was small pieces, snap-frozen in liquid nitrogen, and stored at Ϫ80 C. The blocked by 3% hydrogen peroxide in methanol for 10 min. After 10-min study was approved by the ethics committee of Huddinge University washing, sections were exposed to a 30-min nonimmunoblock using Hospital, Karolinska Institute. Consent was obtained from the donor or diluted normal horse serum (Vectastain, Vector Laboratories, Inc., Bur- their relatives to use the liver tissue for scientific purposes. lingame, CA) in PBS. The sections were incubated with monoclonal anti-FAT/CD36 antibodies (antimurine CD36, Cascade Biosciences), di- cDNA microarray analysis luted 1:500 in PBS containing 2% BSA, at 4 C overnight. Negative con- trols were obtained by replacing the primary antibody with normal The generation (10), use, and analysis (11) of microarrays represent- horse serum. As secondary antibody, a biotinylated horse antimouse ing 3200 cDNAs have been described previously.