David H. Alpersp, and John M. TaylorlJ$$. From the Departments of ... standard rat chow diet (Ralston Purina) ad libitum,,and housed under controlled lighting ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 260, No. 4, Issue of February 25, pp. 1995-1998,1985 0 1985 by The American Society of Biological Chemists, Inc.
Communication
Printed in U.S.A.
solic proteins have been isolated that bind fatty acids, their acyl CoA derivatives, and sterols. Among these proteins is rat liver fatty acid binding protein (FABP’), which is also known as Z protein or sterol carrier protein (1-5). This 14,200-Da polypeptide has been purified, its primary structure defined, and the nucleotide sequence of its mRNA described (1, 3). (Received for publication, August 27, 1984) Liver FABP has been detected in liver, small intestinal epithelium, and adipose tissue using monospecific antisera (1). Jeffrey I. Gordon$$1,Nabil Elshourbagyll, John B. Lowe$**, WarrenS. Liaoll, It hasstriking sequence homology with a 15,100-Da, 132David H. Alpersp, and John M. TaylorlJ$$ amino acid long cytosolic fatty acid binding protein (6) which was called intestinal FABP based on its initial siteof isolation From the Departments of $Biological Chemistry, §Medicine, and **Pathology, Washington University (7). The RNAs that specify liver and intestinal FABP arethe School of Medicine, St. Louis, Missouri 631 10 and (1 The two most abundant mRNA species inadult rat intestinal Gladstone Foundation Laboratories, Department of epithelium, comprising 3 and 2% of translatable RNA sePhysiology, Cardiovascular Research Institute, Uniuersity quences, respectively (8). of California at SanFrancisco, San Francisco, California It is unclear why the intestinal epithelium contains two 94140 cytosolic fatty acid binding proteins. Takahashi et al. (2) have recently prvposed that liver FABP (Z protein) may function We have examined the tissue distribution develand opmental regulation of two low molecular weight cy- as an intracellular albumin. There is indirect evidence that tosolic fatty acid binding proteins.Based on their ini- liver FABP may be involved in the uptake,intracellular tial site of isolation, they have been referred to as livertransport, oxidation, esterification and/or synthesis of fatty andintestinalfatty acidbindingproteins(FABP). acids andtheir CoA derivatives (9-16). Considerably less Cloned cDNAs were used to probe blots of RNAs ex- information is available about the in vivo or in vitro properties tracted froma wide variety of adult rattissues as well of intestinal FABP (6). Low molecular weight cytosolic proas small intestine and liverRNA obtained from fetal, teins thatbind fatty acids and sterols have also been isolated suckling, and weaning animals. The highest concentrafrom a variety of nonhepatic tissues(17,18). In order to better tions of “liver” FABP mRNA were found in small inunderstand the function of rat liver and intestinal FABPs testine and liver. “Intestinal” FABP mRNA is most and their structural relationship to these extra-gastrointesabundantin small bowel RNA whileonlytrace amounts were encountered in liver. Both mRNAs were tinal binding proteins, we have defined the distribution of their mRNAs in a variety of adult rat tissues which exhibit detectable in stomach, colon, pancreas, spleen, lung, heart, testes, adrenal, and brain RNA at 1-8% the different capacities to metabolize lipids. In addition, the concentrations observed in small intestine. Accumula- expression of these genes was examined during development tion of both mRNAs in the small intestinalepithelium and correlated with the remarkable alterations in gut and increases during development. The mRNAs are first liver lipid metabolism which are known to occur during the detectable between the19th and 21st day of gestation. suckling-weaning period (19). They undergo a coordinated3-4-fold increase in concentration within the first h24 after birth. Thereafter, MATERIALS AND METHODS gut levels of intestinal FABP mRNA remain constant Animals-Adult male as well as timed-pregnant Sprague-Dawley during the suckling period while liver FABP mRNA rats were obtained from Charles River Breeding Laboratories, fed a increases an additional 2-fold. Liver FABP mRNA lev- standard ratchow diet (Ralston Purina)ad libitum,,and housed under els are also induced in hepatocytes during the first controlled lighting conditions (12 h on, 12 h off). postnatal day but subsequently do not change during RNA Isolation-Total cellular RNA was isolated from pulverized the suckling and weaning phase, despite marked alter- frozen tissues by guanidine thiocyanate extraction (20). Each RNA ations in hepatic fatty acid metabolism. These obser- sample represented material obtained from pooled fetal, neonatal, or vations support the concept that themajor roleof these adult tissues. Six to 10 animals were used for each developmental entry of lipids into cells and/ stage studied. proteins is to facilitate the Dot and Northern Blot Analyses-For dot blot hybridizations, or their subsequent intracellular transport and compartmentalization. The dataalso raise questions about samples of total cellular RNA were applied with atemplate to nitrocellulose filters at four different concentrations (0.5, 1, 2, and 3 the identityof extragastrointestinal FABPs.
Tissue Specific Expressionand Developmental Regulationof Two Genes Coding for Rat FattyAcid Binding Proteins*
fig) as previously described (21).Yeast RNA was added to these yeast) RNA concentration in samples so that the final total (rat each dot was identical (3 pg). Blots were probed with recombinant plasmids containing full length rat liver (3) and intestinal FABP (6) cDNAs. Plasmid DNA was labeled by nick translation using a kit obtained from Amersham Searle. Hybridization and washing conditions have been detailed elsewhere (3, 6). The relative abundance of FABP mRNAs among the different tissue total cellular RNA preparations was determined by quantitativescanningdensitometry of filter autoradiographs. Each filter contained reference standards of small intestinal RNA representing a 100-fold concentration range. Northern blots of total cellular RNA were constructed and hybridized
+
Recently, several low molecular weight, mammalian cyto* This work was supported by National Institutes of Health Grants AM 31615 and 30292. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisemnt” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ll Fellow of the John A. and George L. Hartford Foundation. $$ Recipient of Research Career Development Award CA00872 from the National Cancer Institute.
1995
The abbreviation used is: FABP, fatty acid binding protein.
Expression of Fatty Acid Binding Protein
1996 Liver FABP cDNA Intestinal
FABP cDNA
RAT
- Hoart -Adronai
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Brain Lung
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- Pancreas - Spioon - Toatoa - Stomach - Kldnoy
- Largo intortine-
0
@e --
0.m
small IntortinoLiver
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Yeast
0.5 I
2
0.5 I 2
3 pg RNA
Genes
hybridization and filter washing had previously been shown to produce no cross-hybridization between the two FABP mRNAs and their cDNAs (3,6). The results are displayed in Fig. 1and summarized inTable I. Liver and intestinalFABP mRNAs were most abundant in small bowel RNA. However, both mRNA sequences were distributed throughout the gastrointestinal tract. Stomach contained approximately 2-4% of small bowellevelswhilecolon contained 5 4 % . Both mRNA sequences were detected in a group of tissues which primarily oxidize fatty acids (heart, kidney, and brain) as well as in a group of tissues that support steroidogenesis (adrenal, testes). Accumulation of both mRNA species in both groups of tissues appeared to increase during growth from120-210 g (Table I). Spleen, pancreas, and lung also gave signals in the dot blot hybridizations whichwere1-4% of the reference small intestinal signals (Table I). Control “dots” of yeast RNA did not react with either FABP cDNA probe (Fig. 1). Despite these similarities in tissue distribution, a striking difference in the concentrations of the mRNAs was noted in liver.Liver FABP mRNAwas abundantly represented in hepatocytes. This was not the casewith intestinal FABP mRNA, which was 25-40-fold less abundant thanliver FABP mRNA, depending onthe age of the animal (see Table I and below).
3 pg RNA
FIG. 1. The distribution of liver and intestinal FABP mRNA in adult rat tissues. Total cellular RNA from a variety of (pooled) -200-g male rat tissues was applied to nitrocellulose filters in the concentrations indicated. Duplicate filters were probed with 32Plabeled cloned double-stranded intestinal or liver FABP cDNA. Hybridization and washing stringencies have been described elsewhere (3,6). An autoradiograph of the filters is shown.
I
I
I
l
l -Origin
TABLE I Relative levelsof intestinal and liver FABP mRNA in adult rat tissues The datashown represent the results of quantitative densitometric tracings of dot blots. Relative mRNA level 120-140-grats
Tissue
Liver FABP FABP FABP FABP
Small intestine Large intestine Stomach Liver Adrenal Testes Kidney Brain Heart Spleen Lung Pancreas
100 6 1.8 73 1.7 1 1.1 1.1 2.5
