ARTICLE |
Correspondence to: Toshimitsu Suzuki, Dept. of Pathology, Fukushima Medical U. School of Medicine, 1-Hikariga-oka, Fukushima, 960-1295, Japan. E-mail: tmt@fmu.ac.jp
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Summary |
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Animal fatty acid synthase (FAS) is a homodimer protein which synthesizes long-chain fatty acids and is rich in liver, brain, breast, and lung. However, the precise cellular localization of FAS in human tissues has not been elucidated. Immunohistochemistry with a new antibody to human FAS revealed that in adult human tissues FAS is distributed mainly in cells with high lipid metabolism (adipocytes, corpus luteum, hepatocytes, sebaceous glands, and Type II alveolar cells), in hormone-sensitive cells (anterior pituitary, apocrine gland, breast, endometrium, prostate, seminal vesicle, and adrenal cortex), and in a subset of epithelial cells of duodenum and stomach, colon absorptive cells, cerebral neurons, basket cells of cerebellum, decidua, uroepithelium, and epidymis. In fetal cells at 20 weeks of gestation, FAS was mainly present in proliferative epithelial cells of the digestive and respiratory systems, proximal renal tubules, adrenocortical cells, and mesenchymal and hematolymphoid cells. Staining was significant in nonproliferating cells, as observed in adult, and in sympathetic ganglion cells, Leidig cells of testis, and Langhans cells of chorionic villi. FAS is maintained in hormone-sensitive cells and/or cells active in lipid metabolism in the adult and is expressed in proliferating cells in the fetus, suggesting active fatty acid synthesis for energy utilization or membrane lipids. (J Histochem Cytochem 48:613622, 2000)
Key Words: fatty acid synthase, cellular localization, fetal tissues, adult tissues
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Introduction |
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ANIMAL FATTY ACID SYNTHASE (FAS: EC 2.3.1.85) (
Here we describe the cellular distribution of FAS in normal human tissues in fetuses, newborns, infants, and adults.
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Materials and Methods |
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Tissues
Normal human tissues were sampled at autopsy from three newborns, one infant, and three adults. For the central nervous system, seven adult brains (four females and three males, 2969 years old) were employed. All cases were free of hormone (insulin and/or corticosteroid) or transfusion treatment. Fetal tissues, placenta, and decidua from 6, 20, 25, and 36 weeks of gestation were obtained from spontaneous abortions or stillbirths. In addition, curetted endometrium, surgically removed gallbladder for treatment of cholecystolithiasis, and extirpated breast for treatment of mammary carcinoma were also employed. Tissues were fixed with 10% neutral formalin and paraffin-embedded. Fetal brains were not included because they had not been processed for routine histological examination.
Antibodies
A polyclonal antibody was produced by immunizing New Zealand White rabbits by SC injections of a synthetic peptide composed of 17 amino acids corresponding to residues 12831299 of human brain FAS, which was coupled with keyhole limpet hemocyanin (Difco; Detroit, MI). The 17 amino-acid peptide is the sequence of the 3' terminus of the predicted human brain FAS gene product (
Western Blotting
Human gastric carcinoma cell line TKGW from IBL was employed because immunostaining of FAS by our antibody was positive in this cell line. A gastric cancer cell line, MKN-1, maintained in our laboratory and negative for FAS immunostaining, was also used. Both were homogenized in lysis buffer containing CHAPS and protease inhibitors (pefabloc, leupeptin, pepstatin, and aprotinin) and centrifuged at 15,000 x g for 30 min. The supernatant was mixed with sample buffer (18.5 ml (1 mol)/liter Tris-HCI, pH 7.8, 15 ml 2-mercaptoethanol, 30 ml glycerol, and distilled water, to 100 ml) in a ratio of 2:1. Protein concentration was determined by the Lowry method, and 10 µg protein was subjected to SDS-PAGE and then blotted onto a nitrocellulose membrane. The membrane was then incubated with 2 µg/ml anti-FAS antibody IgG or FAS antibody absorbed by 5 µg antigen, followed by anti-rabbit goat IgG coupled to horseradish peroxidase (IBL). The peroxidase reaction product was visualized with Konica Immunostain HRP1S-50B (Konica; Tokyo, Japan).
Immunohistochemistry
Immunohistochemical procedures were described previously (
In breast cancer tissues known to be FAS-positive (
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Results |
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Specificity of the FAS Antibody and Validity of Immunostaining
The antibody purified by affinity chromatography reacted with a 270-kD protein in the extracts of liver and TKGW cells but did not react with the extract of MKN-1 by Western blotting, as shown in Fig 1A. The 270-kD protein has the same molecular weight as the monomer of human brain FAS (
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As shown below, the conditions of immunostaining with the FAS antibody were established by immunohistochemistry with human breast cancer tissue, which is known to be FAS-positive (
FAS-positive Cells in Adults
The results with human adult tissues are summarized in Table 1. Strongly FAS-positive cells were mainly nonproliferating, and limited amounts of FAS were detected in areas of active cell turnover, which consisted of cells in the proliferative zone (Ki-67-labeled) of gastroduodenal mucosa, hematopoietic cells in bone marrow, proliferating gland cells, and stromal cells of endometrium. Nonproliferating FAS-positive cells in the CNS were cerebral cortical neurons of the fronto-occipital lobes, mainly in the third to fifth layers (Fig 2A), some neurons in the basal ganglia, hippocampus, paraventriculus, pons and medulla oblongata, basket cells of the cerebellum (Fig 2B), and a few astrocytes. Axons of basket cells that terminate on or attach to Purkinje cell bodies were also positive. Hippocampal basket cells were not labeled. There were no gender differences in the frequency of FAS-positive neurons in cerebral cortices. Cerebellar cortical basket cells showed similar positive staining in the seven brains examined, whereas cerebral cortical neurons exhibited mild fluctuation in the frequency of FAS staining from brain to brain. FAS-positive cells were also found in the digestive system [absorptive cells of the large intestine (Fig 2C ) and the appendix vermiformis and hepatocytes in Zone 1 to Zone 2 (Fig 2D)], some Type II alveolar cells of lung (Fig 3A) also positive for surfactant apoprotein, in the urogenital system [a subset of epithelial cells of the epididymis and seminal vesicle, main prostate glandular cells ( Fig 3B), mucosal and submucosal glands of the prostate, decidualized stromal cells of the endometrium, lutein cells of the ovary, and umbrella cells of the bladder], anterior pituitary cells (Fig 3C) (partly identical with GH and/or ACTH cells), subcutaneous and intra-abdominal fat cells, apocrine gland and sebaceous gland of skin (Fig 3D), tissue mast cells, adrenal zona fasciculata cells, and epithelial cells of ducts and acini of the breast.
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FAS-positive Cells in Fetuses
In the 6-week-old fetus, hepatocytes and hemopoietic cells, including erythroblasts, were positive. Because each visceral organ was easily identified, two 20-week-old fetuses were examined precisely except for their brains. The results are summarized in Table 2. The FAS-positive cells were predominantly those labeled with Ki-67 in areas of cell proliferation, although the Ki-67-labeled cells exceeded the number of FAS-positive cells. Positive cells in the digestive system were basal layer cells of squamous epithelium of tongue and esophagus, nonmucinous salivary gland cells (Fig 4A and Fig 4B), glandular cells of gastrointestinal mucosa, pancreatic acinar cells, and hepatocytes. In the cardiopulmonary system, positive cells were found in the endocardium and tracheobronchial glands, bronchopulmonary epithelium, chondrocytes of tracheobronchial cartilage, and vascular endothelial cells (Fig 4C and Fig 4D). Positive cells of the urogenital system were found in the S-shaped proximal tubules of kidney ( Fig 4E and Fig 4F ) and in the prostate gland. In the endocrine system, anterior pituitary cells, thyroid follicular cells, and subcapsular adrenocortical cells were positive (Fig 4G and Fig 4H). In addition, thymocytes and nodal lymphocytes in the periphery of the tissue were weakly positive. Schwann cells and mesenchymal cells of peripheral nerves in sympathetic ganglia and the Auerbach plexus of the gastrointestinal tract, a few neuroblasts in the adrenal medulla, fibroblasts (especially in the perivertebral region), striated muscle cells of tongue, and hematopoietic cells, including erythroblasts and megakaryocytes, showed cytoplasmic FAS, but the frequency of positive cells was low. However, cardiomyocytes and smooth muscle cells of visceral organs were hardly labeled despite being positive with Ki-67. Some Ki-67-negative cells were FAS-positive in the fetus, but this was limited and consisted of adipocytes, ganglion cells of sympathetic ganglia (proliferating Schwann cells were also positive) and the Auerbach plexus, Leidig cells of testis (proliferating seminiferous tubules were negative) (Fig 5A and Fig 5B), sebaceous gland, urinary tract uroepithelial cells (Ki-67-labeled cells were negative) (Fig 5C and Fig 5D), and the upper layer of adrenocortical cells (Fig 4G ). Tissue mast cells and osteoblasts were also positive. Placenta from the sixth week of gestation or later showed FAS-positive cytotrophoblasts. Syncytiotrophoblasts and other components of the placenta were negative. Tissues of later gestational ages (25 and 36 weeks), newborns at 1 month, infants at 6 months, 10 months, and infants at 3 years had much more limited distributions of FAS-positive cells, almost similar to those of adult tissues. For example, a 1-month-old infant was FAS-negative in proximal tubules of kidney, subcapsular adrenocortical cells, Schwann cells of sympathetic ganglion, and mesenchymal cells (fibroblasts, chondrocytes, endothelial cells), which had ceased to proliferate.
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Discussion |
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Long-chain fatty acids, such as palmitate, are essential constituents of membrane lipids and are also important substrates for cellular energy metabolism. FAS synthesizes mainly palmitate, as stated above. Expression of FAS mRNA in nonfetal human tissues was high in brain, liver, lung, intro-abdominal adipose tissue (
It was reported that decidualized stroma cells in the late secretory phase and decidua of pregnancy were intensely positive for FAS not associated with cell proliferation (
In conclusion, under normal conditions FAS is highly expressed not only in proliferating cells and those with high lipid metabolism but also in hormone-sensitive cells and those with permeability barriers.
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Acknowledgments |
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Supported in part by a Grant-in-Aid for Scientific Research (no. 10670168) from the Ministry of Education, Science, Sports and Culture of Japan.
We thank H. Hiraki for his outstanding help with the immunohistochemical studies.
Received for publication August 18, 1999; accepted January 5, 2000.
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