Analysis of the tissue-specific expression, developmental regulation, and linkage relationships of a rodent gene encoding heart fatty acid binding protein

The rat contains at least three homologous cytosolic proteins that bind long chain fatty acids, termed liver (L-), intestinal (I-), and heart (H-) fatty acid binding protein (FABP). I-FABP mRNA is confined to the gastrointestinal tract while L-FABP mRNA is abundantly represented in hepatocytes as well as enterocytes. We have isolated a rat heart FABP cDNA clone and determined the pattern of H-FABP mRNA accumulation in a wide variety of tissues harvested from late fetal, suckling, weaning, and adult rats. RNA blot hybridizations and primer extension analysis disclosed that the distribution of H-FABP mRNA in adult rat tissues is different from that of I- or L-FABP mRNA. H-FABP mRNA is most abundant in adult heart. This mRNA was also present in an adult slow twitch (type I) skeletal muscle (soleus, 63% of the concentration in heart), testes (28%), a fast twitch skeletal muscle (psoas, 17%), brain (10%), kidney (5%), and adrenal gland (5%). H-FABP mRNA was not detected in adult small intestine, colon, spleen, lung, or liver RNA. Distinct patterns of developmental change in H-FABP mRNA accumulation were documented in heart, placenta, brain, kidney, and testes. Myocardial H-FABP mRNA levels rise rapidly during the 48 h prior to and after birth, reaching peak levels by the early weaning period. The postnatal increase in myocardial H-FABP mRNA concentration and its relative distribution in adult fast and slow twitch skeletal muscle are consistent with its previously proposed function in facilitating mitochondrial beta-oxidation of fatty acids. However, the presence of H-FABP mRNA in brain, a tissue which does not normally significantly oxidize fatty acids in late postnatal life, suggests that H-FABP may play a wider role in fatty acid metabolism than previously realized. Mouse-hamster somatic cell hybrids were utilized to map H-FABP. Using stringencies which did not produce cross-hybridization between L-, I-, and H-FABP DNA sequences, we found at least three loci in the mouse genome, each located on different chromosomes, which reacted with our cloned H-FABP cDNA. None of these H-FABP-related loci were linked to the gene which specifies a highly homologous adipocyte-specific protein termed aP2 or to genes encoding two other members of this protein family, cellular retinol binding protein and cellular retinol binding protein II.(ABSTRACT TRUNCATED AT 400 WORDS)     

Analyzing the structures, functions and evolution of two abundant gastrointestinal fatty acid binding proteins with recombinant DNA and computational techniques

The structures of intestinal and liver fatty acid binding proteins (FABPs) have been determined from an analysis of the nucleotide sequences of cloned cDNAs. The primary translation product of intestinal FABP mRNA contains 132 residues (Mr = 15 124). Liver FABP mRNA encodes a 127 amino acid polypeptide (Mr = 14 273). In vitro co-translational cleavage and translocation assays showed that neither sequence has a cleavable signal peptide or signal peptide equivalent - suggesting that the FABPs do not enter the secretory apparatus but rather are targeted to the cytoplasm. A variety of computational techniques were used to compare the two FABP sequences. The results indicate that liver and intestinal FABP are paralogous homologues. A superfamily of proteins was defined which includes the FABPs, the cellular retinol and retinoic acid binding proteins, the P2 protein of peripheral nerve myelin, and a polypeptide known as 422 whose synthesis is induced during differentiation of 3T3-L1 cells to adipocytes. No sequence homologies were noted between any of these small molecular weight cytosolic proteins and nonspecific lipid transfer protein (sterol carrier protein 2), phosphatidylcholine transfer protein, serum albumin or apolipoprotein AI. The FABPs may have structural features responsible for lipid-protein interactions that are not present in these non-homologous sequences. The distribution of intestinal and liver FABP mRNAs in adult rat tissues and the changes in FABP gene expression which occur during gastrointestinal development support the notion that these proteins are involved in fatty acid uptake, transport and/or compartmentalization. However, differences in tissue distribution and periods of non-coordinate expression during gastrointestinal ontogeny suggest that the two FABPs have distinct functions. The relationship between intestinal and liver FABPs and similar sized cytosolic FABPs isolated from brain, skeletal and cardiac muscle remains unclear. Recombinant DNA techniques combined with comparative sequence analyses offer a useful approach for defining unique as well as general structure-function relationships in this group of fatty acid binding proteins.     

Function and regulation of hepatic and intestinal fatty acid binding proteins

Two structurally different fatty acid binding proteins (FABP) have been isolated from rat liver and small intestinal epithelium. hFABP is a 14 184 Da protein found in abundance in both liver and small intestine, whereas gFABP (15 063 Da) is abundantly present only in small intestine. This review discusses studies which have provided insight into the physiological functions of these proteins. These include analyses of endogenous and exogenous ligand binding to FABP in vitro; examination of the modulating effect of FABP preparations on enzyme activities in vitro; exploration of relationships between alterations in cytosolic FABP content in response to hormonal, pharmacological, and dietary manipulations and changes in the rates of cellular fatty acid uptake and utilization; and studies of hFABP turnover and the mechanisms of FABP regulation. These experiments provide compelling evidence for a broad role of the FABPs in the transport, utilization and cellular economy of free fatty acids in the liver and small intestine, and also in protecting several aspects of cellular function against the modulatory effects of fatty acids, fatty acyl-CoA esters, and other ligands. Studies of FABP regulation also suggest a role in long-term rather than short-term modulation of hepatic fatty acid metabolism and indicate that hFABP and gFABP may perform different functions in the small intestine.     

Isolation and characterization of two fatty acid binding proteins from intestine of Gallus domesticus

1. Two distinct fatty acid binding proteins (FABPs) were isolated and characterized from chicken duodenal mucosa. 2. Molecular weight, functional activity, immunospecificity, mRNA expression, and amino acid composition data for the 14 kDa chicken intestinal FABP was similar, yet not identical, to that of a previously isolated chicken liver FABP. 3. Bound fatty acids were shown to produce isoforms of the 14 kDa intestinal protein but not the larger molecular weight intestinal FABP.     

Fatty acid binding proteins from different tissues show distinct patterns of fatty acid interactions

Fatty acid binding proteins (FABP) form a family of proteins displaying tissue-specific expression. These proteins are involved in fatty acid (FA) transport and metabolism by mechanisms that also appear to be tissue-specific. Cellular retinoid binding proteins are related proteins with unknown roles in FA transport and metabolism. To better understand the origin of these tissue-specific differences we report new measurements, using the acrylodated intestinal fatty acid binding protein (ADIFAB) method, of the binding of fatty acids (FA) to human fatty acid binding proteins (FABP) from brain, heart, intestine, liver, and myelin. We also measured binding of FA to a retinoic acid (CRABP-I) and a retinol (CRBP-II) binding protein and we have extended to 19 different FA our characterization of the FA-ADIFAB and FA-rat intestinal FABP interactions. These studies extend our previous analyses of human FABP from adipocyte and rat FABPs from heart, intestine, and liver. Binding affinities varied according to the order brain approximately myelin approximately heart > liver > intestine > CRABP > CRBP. In contrast to previous studies, no protein revealed a high degree of selectivity for particular FA. The results indicate that FA solubility (hydrophobicity) plays a major role in governing binding affinities; affinities tend to increase with increasing hydrophobicity (decreasing solubility) of the FA. However, our results also reveal that, with the exception of the intestinal protein, FABPs exhibit an additional attractive interaction for unsaturated FA that partially compensates for their trend toward lower affinities due to their higher aqueous solubilities. Thermodynamic potentials were determined for oleate and arachidonate binding to a subset of the FABP and retinoid binding proteins. FA binding to all FABPs was enthalpically driven. The DeltaH degrees values for paralogous FABPs, proteins from the same species but different tissues, reveal an exceptionally wide range of values, from -22 kcal/mol (myelin) to -7 kcal/mol (adipocyte). For orthologous FABPs from the same tissue but different species, DeltaH degrees values were similar. In contrast to the enthalpic dominance of FA binding to FABP, binding of FA to CRABP-I was entropically driven. This is consistent with the notion that FA specificity for FABP is determined by the enthalpy of binding. Proteins from different tissues also revealed considerable heterogeneity in heat capacity changes upon FA binding, DeltaC(p) values ranged between 0 and -1.3 kcal mol(-1) K(-1). The results demonstrate that thermodynamic parameters are quite different for paralogous but are quite similar for orthologous FABP, suggesting tissue-specific differences in FABP function that may be conserved across species.     

Mucosal IgA increase in rats by continuous CLA feeding during suckling and early infancy

The aim of this work was to establish the effect of the cis9,trans11 conjugated linoleic acid (CLA) isomer on mucosal immunity during early life in rats, a period when mucosal immunoglobulin production is poorly developed, as is also the case in humans. CLA supplementation was performed during three life periods: gestation, suckling, and early infancy. The immune status of supplemented animals was evaluated at two time points: at the end of the suckling period (21-day-old rats) and 1 week after weaning (28-day-old rats). Secretory IgA was quantified in intestinal washes from 28-day-old rats by ELISA technique. IgA, TGFbeta, and PPARgamma mRNA expression was measured in small intestine and colon by real time PCR, using Taqman specific probes and primers. IgA mucosal production was enhanced in animals supplemented with CLA during suckling and early infancy: in 28-day-old rats, IgA mRNA expression was increased in small intestine and colon by approximately 6- and 4-fold, respectively, and intestinal IgA protein by approximately 2-fold. TGFbeta gene expression was independent of age and type of tissue considered, and was not modified by dietary CLA. Gene expression of PPARgamma, a possible mediator of CLA's effects was also upregulated in animals receiving CLA during early life. In conclusion, dietary supplementation with CLA during suckling and extended to early infancy enhances development of the intestinal immune response in rats.     

Characterization of crystalline rat liver fatty acid binding protein produced in Escherichia coli

The principal absorptive cell of the rat small intestinal epithelium contains two homologous cytosolic proteins that bind long chain fatty acids. These are known as intestinal and liver fatty acid binding proteins (FABP). While their precise physiological roles have not been defined, they are believed to represent a multifunctional cytosolic transport system that is involved in the trafficking of exogenous lipids to sites of metabolic processing. 13C NMR studies have revealed differences in their fatty acid binding stoichiometries, binding mechanisms, and the ionization properties of bound fatty acids. To understand the functional differences, liver FABP has been crystallized for eventual comparison with the known crystal structure of intestinal FABP. The lattice type is trigonal with unit cell dimensions of a = b = 84.1 A and c = 44.2 A. The space group as determined by examination of the Patterson symmetry is either P3(1)21 or P3(2)21.     

The tissue-specific expression and developmental regulation of two nuclear genes encoding rat mitochondrial proteins. Medium chain acyl-CoA dehydrogenase and mitochondrial malate dehydrogenase

To study the regulation of nuclear genes which encode mitochondrial enzymes involved in oxidative metabolism, absolute levels of mRNA encoding rat medium chain acyl-CoA dehydrogenase (MCAD) and rat mitochondrial malate dehydrogenase (mMDH) were determined in developing and adult male rat tissues. MCAD mRNA is expressed in a variety of adult male tissues with highest steady state levels in heart, adrenal, and skeletal muscle and lowest levels in brain, lung, and testes. In comparison, steady state levels of mMDH mRNA in adult male rat tissues were similar to those of MCAD mRNA in heart, small intestine, adrenal, and skeletal muscle but markedly different in brain, stomach, and testes. Thus, the steady-state levels of MCAD and mMDH mRNA are highest in adult tissues with high energy requirements. Dot blot analysis of RNA prepared from late fetal, suckling, and weaning rat heart, liver, and brain demonstrated the presence of MCAD and mMDH mRNA during the fetal period in all three tissues. Both MCAD and mMDH mRNA levels increased 2-2.5-fold at birth followed by a decline during the first postnatal week in heart and liver. The patterns of accumulation of these mRNAs in heart and liver during the weaning and early adult periods were also similar, although the absolute levels were significantly different. Brain MCAD mRNA levels were consistently low (less than 0.1 pg/micrograms total cellular RNA) throughout the developmental stages. However, brain mMDH mRNA levels exhibited a marked increase during the weaning period, reaching a peak concentration which is higher than the level of mMDH mRNA in heart and liver at any point during development. These results indicate that the level of expression of the nuclear genes encoding MCAD and mMDH is tissue-specific and developmentally regulated. The patterns of MCAD and mMDH mRNA accumulation parallel the changes in energy metabolism which occur during development and among adult tissues.     

Developmental changes in the expression of genes involved in cholesterol biosynthesis and lipid transport in human and rat fetal and neonatal livers

Cloned cDNAs encoding a number of enzymes involved in cholesterol biosynthesis as well as extracellular and intracellular lipid transport were used to compare the developmental maturation of these biologic functions in the fetal and neonatal rat and human liver. The results of RNA blot hybridization analyses indicate that steady-state levels of rat HMG-CoA synthase, HMG-CoA reductase and prenyl transferase mRNAs are highest in late fetal life and undergo precipitous (up to 80-fold) co-ordinate reductions immediately after parturition. These changes reflect the ability of the fetal rat liver to produce large quantities of cholesterol as well as the repression of this function during the suckling period in response to exogenous dietary cholesterol. Striking co-ordinate patterns of HMG-CoA synthase, reductase and prenyl-transferase mRNA accumulation were also observed in four extrahepatic rat tissues (brain, lung, intestine and kidney) during the perinatal period. The concentrations of all three mRNAs in the 8-week-old human fetal liver are similar to those observed throughout subsequent intrauterine development with less than 2-fold changes noted between the 8th through 25th weeks of gestation. Analysis of the levels of human apo AI, apo AII, apo B and liver fatty acid binding protein mRNAs during this period and in newborn liver specimens also indicated less than 2-3-fold changes. These observations suggest that the 8-week human liver has achieved a high degree of biochemical differentiation with respect to functions involved in lipid metabolism/transport which may be comparable to that present in 19-21 day fetal rat liver. Further analysis of human and rat fetal liver RNAs using cloned cDNAs should permit construction of a developmental time scale correlating hepatic biochemical differentiation to be constructed between these two mammalian species.     

Comparison of the tissue-specific expression and developmental regulation of two closely linked rodent genes encoding cytosolic retinol-binding proteins

Cellular retinol-binding protein (CRBP) and cellular retinol-binding protein II (CRBP II) are two highly homologous cytoplasmic proteins that bind all-trans-retinol. We have recently demonstrated that the mouse genes encoding CRBP and CRBP II are closely linked on chromosome 9 and that both human genes are located on chromosome 3 (Demmer, L.A., Birkenmeier, E.H., Sweetser, D.A., Levin, M.S., Zollman, S., Sparkes, R.S., Mohandas, T., Lusis, A.J., and Gordon, J.I. (1987) J. Biol. Chem. 262, 2458-2467). We have now used RNA blot hybridization analysis to assess the degree to which these genes are coordinately expressed in fetal, suckling, weaning, and adult rat tissues. Both genes exhibit different developmental patterns of expression in liver, intestine, lung, kidney, testes, and placenta. In the intestine, CRBP mRNA was detected during the 16th day of gestation--prior to the development of a well-differentiated absorptive epithelium--and remained essentially unchanged throughout the peri- and postpartum periods. By contrast, the pattern of intestinal CRBP II mRNA accumulation closely parallels the times of first appearance, and subsequent proliferation, of the intestinal absorptive columnar epithelium, supporting the hypothesis that CRBP II is involved in the intestinal uptake or intracellular trafficking of this hydrophobic vitamin. In the fetal liver, both genes were expressed by gestational day 16. Whereas the concentration of hepatic CRBP mRNA increased markedly during the suckling and early weaning periods, CRBP II mRNA levels fell abruptly immediately after birth. These peripartum changes were not paralleled by remarkable alterations in the steady state levels of hepatic retinol. Marked changes in the expression of CRBP in the liver and of CRBP II in the intestine were also documented in pregnant and lactating female rats. These differences in CRBP/CRBP II gene expression strongly suggest that their proteins serve different physiological functions. The peripartum liver may provide a useful model for dissecting the relative roles played by these homologous proteins in retinoid metabolism as well as the factors which modulate activation and repression their genes.     

Tissue specific expression of p422 protein, a putative lipid carrier, in mouse adipocytes

The differentiation of 3T3-L1 preadipocytes leads to the expression of a new protein, p422, and its mRNA. This protein has 70% and 20-30% amino acid sequence homology to myelin P2 and the fatty acid binding proteins of liver and intestine, respectively. Investigation of the distribution in mouse tissues of p422 protein by immunoblotting and of p422 mRNA by cDNA hybridization indicates that they are expressed only in adipose tissue. Liver and intestinal fatty acid binding protein mRNA's were not detectable in mouse adipose tissue or in 3T3-L1 adipocytes. It is suggested that p422 functions as an adipocyte fatty acid binding protein.     

Expression of differentiated functions in the developing porcine small intestine

Heterologous cDNA clones were used as hybridization probes to define the temporal expression of intestinal functions during fetal and postnatal development in the pig. Northern hybridization analysis revealed the presence of the mRNAs for the cellular retinol binding protein CRBP II, for the digestive enzyme aminopeptidase N, and for the microvillar proteins villin and ezrin in the small intestine of both weaned and 40-day fetal pigs. The presence of these mRNAs suggests that at the end of the first third of gestation the pig fetal intestine is already exhibiting some characteristics of a differentiated epithelium. The mRNAs for the two fatty acid-binding proteins I-FABP and L-FAPB, both involved in the metabolism of long chain fatty acids, were detected only in the intestinal mRNA extracted from weaned animals, while that for the cellular retinol-binding protein CRBP I was expressed only in the fetal tissue. The temporal limits of expression of intestinal genes in the pig epithelium seem therefore more easily defined than in other experimental animals with shorter times of fetal development. To isolate pig genes expressed at different developmental stages during intestinal epithelial cell differentiation, a cDNA library was constructed from poly(A) + RNA extracted from mature pig intestine. This library was employed in the isolation of clones encoding CRBP II and L-FABP. The nucleotide sequence of the two pig cDNA clones was determined, and the sequences of the deduced proteins compared with their homologues from other species. The results of this analysis showed that the two pig clones share a high level of homology with human and rat homologues both at the DNA and at the protein level.     

Effect of liver fatty acid binding protein on fatty acid movement between liposomes and rat liver microsomes.

Although movement of fatty acids between bilayers can occur spontaneously, it has been postulated that intracellular movement is facilitated by a class of proteins named fatty acid binding proteins (FABP). In this study we have incorporated long chain fatty acids into multilamellar liposomes made of phosphatidylcholine, incubated them with rat liver microsomes containing an active acyl-CoA synthetase, and measured formation of acyl-CoA in the absence or presence of FABP purified from rat liver. FABP increased about 2-fold the accumulation of acyl-CoA when liposomes were the fatty acid donor. Using fatty acid incorporated into liposomes made either of egg yolk lecithin or of dipalmitoylphosphatidylcholine, it was found that the temperature dependence of acyl-CoA accumulation in the presence of FABP correlated with both the physical state of phospholipid molecules in the liposomes and the binding of fatty acid to FABP, suggesting that fatty acid must first desorb from the liposomes before FABP can have an effect. An FABP-fatty acid complex incubated with microsomes, in the absence of liposomes, resulted in greater acyl-CoA formation than when liposomes were present, suggesting that desorption of fatty acid from the membrane is rate-limiting in the accumulation of acyl-CoA by this system. Finally, an equilibrium dialysis cell separating liposomes from microsomes on opposite sides of a Nuclepore filter was used to show that liver FABP was required for the movement and activation of fatty acid between the compartments. These studies show that liver FABP interacts with fatty acid that desorbs from phospholipid bilayers, and promotes movement to a membrane-bound enzyme, suggesting that FABP may act intracellularly by increasing net desorption of fatty acid from cell membranes.     

The fatty liver dystrophy (fld) mutation. A new mutant mouse with a developmental abnormality in triglyceride metabolism and associated tissue-specific defects in lipoprotein lipase and hepatic lipase activities

An autosomal recessive mutation, termed fatty liver dystrophy (fld), can be identified in neonatal mice by their enlarged and fatty liver (Sweet, H. O., Birkenmeier, E. H., and Davisson, M. T. (1988) Mouse News Letter 81, 69). We have examined the underlying metabolic abnormalities in fld/fld mice from postnatal days 3-40. Serum and hepatic triglyceride levels were elevated 5-fold in suckling fld/fld mice compared to their +/? littermates but abruptly resolved at the suckling/weaning transition. Blot hybridization analysis of liver and intestinal RNAs revealed a liver-specific increase in apolipoprotein (apo) A-IV and C-II mRNA concentrations (100- and 6-fold, respectively) that was limited to the suckling and early weaning stages in fld/fld mice. Resolution of these differences during the weaning period could not be delayed by prolonging suckling to the 20th postnatal day nor could the mutant phenotype be elicited in young adult animals with a high fat diet. Lipoprotein lipase (LPL) activity was reduced 16-fold in the white adipose tissue of fld/fld mice until the onset of weaning. Heart activity was decreased less than 2-fold, but there were no deficits in brown adipose tissue or liver. Hepatic lipase (HL) mRNA levels and activity were significantly reduced in fld/fld livers and sera, respectively, during the suckling period. Mapping studies show the fld locus to be distinct from loci encoding LPL, HL, and apoA-IV, and those responsible for the combined lipase deficiencies in cld/cld and W/Wv mice. These data suggest that the fld mutation is associated with developmentally programmed tissue-specific defects in the neonatal expression of LPL and HL activities and provide evidence for a new regulatory locus which affects these lipase activities. This mutation could serve as a useful model for (i) analyzing the homeostatic mechanisms controlling lipid metabolism in newborn mice and (ii) understanding and treating certain inborn errors in human triglyceride metabolism.     

Diversity of fatty acid-binding protein structure and function: studies with fluorescent ligands

The mammalian fatty acid-binding proteins (FABP) are localized in many distinct cell types. They bind long chain fatty acidsin vitro, however, their functions and mechanisms of actionin vivo remain unknown. The present studies have sought to understand the relationships among these proteins, and to address the possible role of FABP in cellular fatty acid traffic. A series of anthroyloxy-labeled fluorescent fatty acids have been used to examine the physicochemical properties of the fatty acid-binding sites of different members of the FABP family. The fatty acid probes have also been used to study the rate and mechanism of fatty acid transfer from different FABP types to phospholipid membranes. The results of these studies show a number of interesting and potentially important differences between FABP family members. An examination of adipocyte and heart FABP (A- and H-FABP) shows that their fatty acid-binding sites are less hydrophobic than the liver FABP (L-FABP) site, and that the bound ligand experiences less motional constraint within the A- and H-FABP binding sites than within the L-FABP binding site. In keeping with these differences in structural properties, it was found that anthroyloxy-fatty acid transfer from A- and H-FABP to membranes is markedly faster than from L-FABP. Moreover, the mechanism of fatty acid transfer was found to be similar for the highly homologous logous A- and H-FABP, whereby transfer to phospholipid membranes appears to occur via transient collisional interactions between the FABP and membranes. Transfer of fatty acids from L-FABP, in contrast, occurs via an aqueous phase diffusion mechanism. Other studies utilized fluorescent fatty acid and monoacylglycerol derivatives to compare how the two FABP which are present in high abundance in the proximal small intestine interact with the two major products of dietary triacylglycerol hydrolysis. The results showed that whereas L-FABP binds both fatty acid and monoacylglycerol derivatives, intestinal FABP (I-FABP) appears to bind fatty acid but not monoacylglycerol. In summary, studies with fluorescent ligands have demonstrated unique properties for different FABP family members. A number of these differences appear to correlate with the degree of primary sequence homology between the proteins, and suggest functional diversity within the FABP family.Abbreviations FABP Fatty Acid-Binding Protein - L-FABP Liver FABP - H-FABP Heart FABP - A-FABP Adipocyte FABP - I-FABP Intestinal FABP - AOffa n-(9-anthroyloxy)fatty acid - MG Monoacylglycerol - NBD-PE N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine     

Elevated iron absorption in the neonatal rat reflects high expression of iron transport genes in the distal alimentary tract

Intestinal iron absorption is extremely high in neonatal mammals but falls rapidly to adult levels following weaning. The aim of this study was to investigate the molecular basis of this elevated neonatal absorption using the rat as an experimental model. RNA was extracted from various sections of the intestine of 10-, 15-, 20-, 25-, and 300-day-old rats and the expression of the genes encoding DMT1 (Slc11a2), ferroportin (Slc40a1), Cybrd1 (Cybrd1), and hephaestin (heph) determined by ribonuclease protection assay. The hepatic expression of Hamp was studied at the same ages. Iron absorption was examined by following (59)Fe uptake in both whole animals and in isolated intestinal loops. Slc11a2, Slc40a1, and Cybrd1 mRNAs were highly expressed in all regions of the small intestine and colon studied in suckling rats. However, after weaning, when iron absorption declined significantly, strong expression was retained only in the duodenum. No change in hephaestin mRNA occurred in any part of the digestive tract. In the distal small intestine and colon, Slc40a1 expression most closely followed the change in absorption that occurred after weaning. Hamp expression was low during the neonatal period and increased to adult levels following weaning. Our results suggest that the distal small intestine and colon contribute significantly to the high intestinal iron absorption seen in neonatal animals and that this reflects increased expression of the iron transporters, particularly Slc40a1.     

Tissue-specific expression of genes encoding apolipoprotein E and apolipoprotein A-I in rabbits

We determined the site of synthesis of apolipoprotein (apo) E and apo-A-I in rabbit by measuring in vitro translational activity of their mRNAs from the liver and from the intestine. Poly(A+) RNA isolated from liver and intestinal epithelium of rabbits fed either a chow diet or a cholesterol-rich diet was translated in vitro in the rabbit reticulocyte lysate system using [35S] methionine as the labeled precursor. Newly synthesized apolipoproteins were immunoprecipitated with specific antisera and quantitated after electrophoresed on 10% polyacrylamide slab gels in the presence of 0.2% sodium dodecyl sulfate. The levels of liver apo-E and apo-A-I mRNAs from chow-fed rabbits are 0.41 and 0.002% of total translatable mRNA, respectively. The level of liver apo-A-I mRNA in the rabbit is approximately 500-fold lower than the reported level of apo-A-I mRNA in rat and human livers. Rabbit intestinal apo-E and apo-A-I mRNAs levels are 0.0036 and 0.67%, respectively. Our results indicate that in rabbits apo-E is synthesized primarily in the liver and that apo-A-I is synthesized primarily in the intestine. When rabbits are fed a cholesterol-rich diet, liver and intestinal apo-E in mRNA levels and intestinal apo-A-I mRNA levels are not changed. In contrast, the liver apo-A-I mRNA level increases 5-fold in response to the cholesterol-rich diet. However, because the intestinal liver apo-A-I mRNA level is so low, the 5-fold induction only increases liver mRNA levels to 2.7% of the corresponding intestinal apo-A-I mRNA level.