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Recently, we demonstrated that adipose tissue plays an important role in retinol storage and retinol-binding protein (RBP) synthesis. Our data suggested that RBP expression in adipose tissue is dependent on the state of adipocyte differentiation. To examine this possibility, we explored the differentiation-dependent expression of RBP using BFC-1 beta preadipocytes, which can be stimulated to undergo adipose differentiation. Total RNA was isolated from undifferentiated (preadipocytes) and differentiated (adipocytes) BFC-1 beta cells and analyzed by Northern blotting. RBP mRNA was not detected in the preadipocytes, but considerable RBP mRNA was present in differentiated BFC-1 beta cells. In BFC-1 beta cells, induced to differentiate with insulin and thyroid hormone, RBP mRNA was first detected after 4 days, reached a maximum level by day 10, and remained at this maximum level for at least 2 more days. Cellular retinol-binding protein was expressed at low levels in the BFC-1 beta preadipocytes and the level of expression increased for 6 days after induction to differentiate and slowly declined on later days. Neither the maximum level of RBP expression nor the day on which this level was reached was influenced by the level of retinol provided in the BFC-1 beta culture medium. BFC-1 beta cells secreted newly synthesized RBP into the culture medium at a rate of 43 +/- 14 ng RBP/24 h/10(6) adipocytes. When the BFC-1 beta adipocytes were provided 1.0 microM retinol in the medium, they accumulated the retinol and synthesized retinyl esters. These studies with BFC-1 beta cells confirm that RBP synthesis and secretion and retinol accumulation are intrinsic properties of differentiated adipocytes. Furthermore, they suggest that RBP and cellular retinol-binding protein gene expression are regulated as part of a package of genes which are modulated during adipocyte differentiation.

Adipose tissue has been reported to contain relatively high levels of the specific mRNA for retinol-binding protein (RBP) (Makover A., Soprano, D.R., Wyatt, M. L., and Goodman, D.S. (1989) J. Lipid Res. 30, 171-180). Studies were conducted to explore retinoid and retinoid-binding protein storage and metabolism in adipose tissue. In these studies, we measured RBP and cellular retinol-binding protein (CRBP) mRNA levels and retinoid levels in 6 adipose depots in male rats. Total RNA was isolated from inguinal, dorsal, mesenteric, epididymal, perinephric, and brown adipose tissue, and average RBP and CRBP mRNA levels were determined by Northern blot analysis. The relative levels of RBP mRNA in these 6 anatomically different adipose depots averaged, respectively, 6.3, 6.7, 16, 34, 37, and 21% of the level in a rat liver RNA standard. Retinoid levels in the 6 depots were similar and averaged approximately 6-7 micrograms of retinol eq/g of adipose tissue. Since adipose tissue contains several cell types, the cellular localizations of RBP and CRBP expression and retinoid storage were examined. RNA was prepared from isolated rat adipocytes and stromal-vascular cells. Cellular levels of the mRNAs for RBP, CRBP, apolipoprotein E (apoE), lipoprotein lipase, adipocyte P2, and adipsin were measured by Northern blot analysis. RBP was expressed almost exclusively in the adipocytes and only weakly in the stromal-vascular cells. Both CRBP and apoE mRNA levels were relatively high in the stromal-vascular cell preparations and only very low mRNA levels were found in the adipocytes. Lipoprotein lipase, adipsin, and adipocyte P2 mRNAs were found in substantial levels in both the adipocytes and stromal-vascular cells, but with higher levels present in the adipocytes. Cultured adipocytes synthesized RBP protein and secreted it into the medium. Only adipocytes (not stromal-vascular cells) contained retinol, at levels between 0.65-0.8 micrograms of retinol eq/10(6) cells. These studies demonstrate that adipocytes store retinoid and synthesize and secrete RBP, and suggest that rat adipocytes may be dynamically involved in retinoid storage and metabolism.

Studies were conducted to determine if the expression of the gene for retinol-binding protein (RBP) and/or transthyretin (TTR) could be induced upon differentiation of F9 teratocarcinoma cells to either visceral endoderm or parietal endoderm. Both TTR mRNA and RBP mRNA were undetectable in the undifferentiated F9 stem cells and in F9 cells differentiated to parietal endoderm. However, TTR mRNA and RBP mRNA were both detected in F9 cell aggregates differentiated to embryoid bodies (which contain visceral endoderm-like cells) by treatment of the aggregates in suspension with retinoic acid. TTR mRNA was observed at 3 days, and RBP mRNA at 5 days, after treatment of the F9 cell aggregates with retinoic acid. Both TTR mRNA and RBP mRNA were found to be specifically localized by in situ hybridization in the outer layer of cells (the visceral endoderm-like cells) of the embryoid bodies. Finally, synthesis and secretion of both RBP and TTR by F9 cell embryoid bodies was demonstrated by specific immunoprecipitation of each newly synthesized protein from the culture medium. These data thus demonstrate the production and presence of RBP mRNA and TTR mRNA, and the synthesis and secretion of RBP and TTR, by F9 cell embryoid bodies (specifically by visceral endoderm-like cells). This finding suggests that these two proteins may be synthesized by rodent embryos extremely early in embryonic development.

Human platelet-derived growth factor (PDGF) has been previously shown to stimulate low density lipoprotein (LDL) receptor activity in cultured cells. Studies were conducted to delineate in detail the effects of PDGF on the LDL receptor pathway in normal human fibroblasts and to explore relationships between the effects of PDGF on LDL metabolism, on cholesterol metabolism, and on DNA synthesis. Increasing concentrations of PDGF stimulated parallel increases in both DNA synthesis and 125I-LDL cell surface binding. The effect of PDGF was due entirely to an increase (up to 4.3-fold) in the number of receptor sites per cell, and not to a change in receptor affinity (Kd approximately 2.0 nM). Parallel PDGF concentration-dependent increases in 125I-LDL binding, internalization, and degradation at 37 degrees C were observed. The results indicate that PDGF-stimulated cells metabolize receptor-bound LDL in a manner that is identical with that seen with quiescent cells. A single study with highly purified PDGF demonstrated that it was PDGF itself, and not some other component in the partially purified PDGF preparation used in most of this work, that was responsible for the observed effects. Studies were conducted on the effects of PDGF on hydroxymethylglutaryl CoA reductase activity, on cholesterol esterification, and on down-regulation by LDL of the LDL receptor. These studies indicated that LDL cholesterol taken into the PDGF-stimulated cell via the receptor pathway, appeared to become available normally and to have metabolic effects within the cell similar to those seen in quiescent cells. Fibroblasts from subjects with familial hypercholesterolemia showed a normal mitogenic response to PDGF, despite the absence or near absence of an effect on the LDL receptor pathway. Finally, studies were also conducted with endothelial cell-conditioned medium (ECCM), used as a source of the endothelial cell-derived growth factor. ECCM was similar to PDGF in stimulating LDL binding, but differed strikingly from PDGF in that the degradation of internalized LDL was inhibited. As a result, ECCM-treated cells did not effectively increase cholesterol esterification or suppress hydroxymethylglutaryl CoA reductase activity when LDL was present. These findings with substances produced by endothelial cells may have important implications for atherogenesis.

The main retinoids and some binding proteins and enzymes involved in retinol metabolism have been quantified in different types of rat liver cells. Hepatic perisinusoidal stellate cells contained 28-34 nmol of retinoids/10(6) cells, and parenchymal liver cells contained 0.5-0.8 nmol of retinoids/10(6) cells, suggesting that as much as 80% of more of total liver retinoids might be stored in stellate cells with the rest stored in parenchymal cells. Isolated endothelial cells and Kupffer cells contained very low levels of retinoids. More than 98% of the retinoids recovered in stellate cells were retinyl esters. Isolated parenchymal and stellate cell preparations both contained considerable retinyl palmitate hydrolase and acyl-CoA:retinol acyltransferase activities. Parenchymal cells accounted for about 75-80% of the total hepatic content of these two enzyme activities, with the rest located in stellate cells. On a cell protein basis, the concentrations of both of these activities were much greater in stellate cells than in parenchymal cells. In contrast, cholesteryl oleate and triolein hydrolase activities were fairly evenly distributed in all types of liver cells. Large amounts of cellular retinol binding proteins were also found in parenchymal and stellate cells. Although parenchymal cells accounted for more than 90% of hepatic cellular retinol binding protein, the concentration of the protein in stellate cells (per unit protein) was 22 X greater than that in parenchymal cells. Stellate cells were also enriched in cellular retinoic acid binding protein. Thus, both parenchymal and stellate cells contain substantial amounts of retinoids and of the enzymes and intracellular binding proteins involved in retinol metabolism. Stellate cells are particularly enriched in these several components.

Relationships between mevalonate and DNA synthesis were explored in quiescent human fibroblasts stimulated with human platelet-derived growth factor (PDGF). Studies of others have indicated that mevalonate, or a product of mevalonate other than cholesterol, is essential for DNA replication. The present studies were designed to determine whether there was a critical time in the cell cycle when mevalonate was necessary for later DNA synthesis to occur. Compactin and mevinolin, inhibitors of hydroxymethylglutaryl CoA reductase, were employed to block both the synthesis of mevalonate and of DNA. Compactin inhibited the sharp peak of DNA synthesis which occurs in cells 24 h after PDGF addition in a concentration-dependent manner. This suppression of DNA synthesis was not prevented by low density lipoprotein but was fully reversed by mevalonate. Compactin inhibited DNA synthesis when the inhibitor was present during the time interval 10-20 h after PDGF addition. Its presence only in the interval before 10 h or after 20 h had no effect. Conversely, mevalonate could fully overcome the compactin block in DNA synthesis when present during the period of from 10-20 h after PDGF addition. Mevalonate present only before 10 h or after 20 h had no effect. When mevalonate was added to mevinolin-blocked cells for the interval 10-15 h after PDGF, the mevinolin block of DNA synthesis was 68% overcome; in contrast, only 20% of the reversal of the mevinolin block was seen when mevalonate was added from 15-20 h. Addition of mevalonate for only the 2-h interval of from 10-12 h after PDGF overcame the mevinolin block of DNA synthesis (assayed at 24 h) by 50%. The results show that there is a critical time period, several h before S phase, when PDGF-stimulated cells require mevalonate in order for DNA synthesis to proceed at 24 h. This critical period comprised the interval of approximately 10-20 h after PDGF addition and especially the early part of this interval.

Studies were conducted to explore in rats the role of retinol in the regulation of the synthesis and secretion of retinol-binding protein (RBP) by the visceral yolk sac compared to the liver. Previous studies have shown that in retinol deficiency, hepatic RBP secretion is specifically inhibited, whereas hepatic RBP synthesis rate is unchanged. Retinol-depleted, retinoic acid-supplemented female rats were mated, and maternal liver, fetal liver, and visceral yolk sac were obtained at 14 days of gestation (retinol-depleted group). A group of identically treated, retinol-depleted rats were repleted with retinol on the 14th day of gestation, and the same tissues were collected 6 h later (retinol-repleted group). Normal female rats were used as controls. RBP was assayed by radioimmunoassay and RBP mRNA levels by RNase protection assay using a rat RBP cDNA clone. RBP levels in the visceral yolk sac were elevated 10-fold in the retinol-depleted as compared to the control rats and had declined to near normal values in the retinol-repleted animals. The relative levels of RBP mRNA in the visceral yolk sac were very similar in all three groups of rats. Thus, as in the liver, in the visceral yolk sac retinol deficiency inhibits RBP secretion without altering RBP mRNA levels. In the visceral yolk sac, as in the liver, retinol status appears to regulate RBP secretion specifically, without affecting the rate of RBP biosynthesis.

Studies were conducted to ascertain if transthyretin mRNA was present in extrahepatic tissues of the rat. A trnasthyretin cDNA clone was isolated from a lambda gt11 human liver cDNA library by antibody screening and its identity was confirmed by nucleotide sequence analysis. This transthyretin cDNA clone was used to survey poly(A+) RNA isolated from 12 different rat tissues for transthyretin mRNA by Northern blot analysis. The liver contained the highest level of transthyretin mRNA and this level was not altered by the vitamin A status of the rat. A significant amount of transthyretin mRNA was found in the brain (30% of the level of the liver) which was localized in specific regions of the brain. In addition, detectable levels of transthyretin mRNA (1% to 2% of that of the liver) were observed in the stomach, heart, skeletal muscle, and spleen. Translation of brain poly(A+) RNA in rabbit reticulocyte lysates and immunoprecipitation of the translation products with anti-transthyretin antiserum resulted in a protein band of the same size as liver pre-transthyretin. Liver pre-transthyretin was processed by the cotranslational addition of dog pancreas microsomal membranes to a protein that migrated coincidentally with monomeric serum transthyretin. These data suggest that transthyretin in the brain and the cerebrospinal fluid results from de novo synthesis and that transthyretin may play a significant physiological function, as yet unknown, within the nervous system.

Transthyretin (TTR) is involved in the plasma transport of both retinol and thyroid hormones. TTR is synthesized in the liver and choroid plexus, and in small amounts in several other tissues. A study was conducted to determine the tissue sites of degradation and turnover of TTR in the rat. The study employed TTR labeled with tyramine cellobiose (TC) and the trapped ligand method. Samples of purified rat TTR were labeled either with 125I-TC or directly with 131I. A mixture of the two labeled TTRs was injected intravenously into six rats. Blood samples were collected via a venous catheter for kinetic (turnover) analysis. After 24 or 48 h, the rats were killed, and 23 different tissues/organs were assayed as possible sites of TTR degradation. Derivatization of TTR with TC did not appreciably alter TTR plasma kinetics. Plasma turnover data were best fit by a three-pool model. The mean fractional turnover of plasma TTR was 0.15/h, and of total body TTR 0.04/h. The major sites of TTR degradation were the liver (36-38% of total body TTR degradation, almost all in hepatocytes), muscle (12-15%), and skin (8-10%). Tissues that were sites of 1-8% of body TTR degradation included kidneys, adipose tissue, testes, and the gastrointestinal tract. Less than 1% of total TTR degradation occurred in the other tissues examined. A second study was conducted in which labeled TTR was injected intraventricularly into the cerebrospinal fluid in order to explore the degradation of TTR of choroid plexus origin. The kinetics of the appearance and disappearance of such labeled TTR in plasma were physiologically reasonable, with an estimated turnover of cerebrospinal fluid TTR of the order of 0.33/h. The major tissue sites of degradation of labeled TTR injected into cerebrospinal fluid and into plasma were approximately the same. No specific degradation of TTR was found in the nervous system tissues. The most active organs of TTR catabolism, per gram wet weight, were liver and kidneys. These studies demonstrate that many tissues participate in TTR turnover and degradation; the studies provide quantitative information about the tissue sites of TTR catabolism.

Studies were conducted to explore the role of retinol in the control of the rate of synthesis of plasma retinol-binding protein (RBP) in the liver of the rat. Previous studies have shown that nutritional retinol status strongly influences RBP secretion from the liver cell. Both the in vivo relative rate of RBP synthesis and the in vitro translatable level of RBP-specific mRNA were examined in normal vitamin A, retinol-depleted, and retinol-repleted rats. The relative rate of RBP synthesis was estimated by measuring the extent of incorporation of [3H]leucine, [3H]lysine, and [3H]phenylalanine after a 12-min pulse label into immunoprecipitable RBP, relative to the incorporation of these amino acids into total liver trichloroacetic acid-precipitable protein. The level of translatable RBP-specific mRNA was quantitated in vitro by translation of rat liver poly(A+)RNA in the rabbit reticulocyte lysate protein-synthesizing system. The amount of newly synthesized RBP was determined relative to the amount of newly synthesized total protein. Both the relative rate of RBP synthesis (approximately 0.26%) and the translatable level of RBP-specific mRNA (approximately 0.14%) were found to be constant regardless of the retinol status of the rats. These results indicate that retinol, the molecule that RBP specifically binds and transports, does not appear to control the rate of synthesis of RBP or the translatable level of RBP-specific mRNA in the liver of the rat. Regulation of plasma RBP levels by retinol must be exercised at a locus beyond that of RBP synthesis.

Studies were conducted to explore the effects of differences in retinoid nutritional status and of sex on the tissue distribution and levels of cellular retinol-binding protein (CRBP) and of cellular retinoic acid-binding protein (CRABP) in the rat. Sensitive and specific radioimmunoassays were developed and employed to measure the levels of both CRBP and CRABP. Four groups of six male rats each were fed experimental diets that differed greatly in the amount and kind of retinoids provided, but were otherwise identical. These groups were comprised of rats that were normal controls, retinoid-deficient, retinoic acid-fed, and excess retinol-fed. A fifth group of six female rats was fed the control diet. Immunogens identical with rat testis CRBP and CRABP, as assessed by radioimmunoassay displacement curves, were found in every rat tissue examined (21 tissues in males, 18 in females). The highest levels of CRBP were found in the proximal portion of the epididymis, the liver, and kidney. The highest levels of CRABP were found in the seminal vesicles, vas deferens, and skin. A significant (p less than 0.01) inverse relationship was found between CRBP and CRABP levels in the different tissues of the male reproductive tract. In both males and females, CRBP levels were highest in the gonads and proximal portion of the reproductive tract and decreased distally, whereas the opposite was true for CRABP. Retinoid-deficient rats showed reduced tissue levels of CRBP; thus, tissue CRBP levels are influenced by diet and retinoid availability. No differences in tissue CRBP levels were found in the rats fed the control, the retinoic acid, or the excess retinol diets. Female control rats had higher CRBP levels than male controls in 4 of 15 tissues compared (liver, lung, thymus, and fat). In contrast, tissue CRABP levels showed no diet- or sex-dependent differences. Only in one tissue, the skin, were differences observed (lower CRABP in retinoid-deficient and in female rats). Thus, CRABP metabolism and levels appear to be minimally influenced by the amount or kind of retinoid ligand available or by sex.

A study was performed to identify the first translated product of messenger RNA for retinol-binding protein (RBP), the specific plasma transport protein for vitamin A. Poly(A)+RNA was isolated from rat liver and translated in the rabbit reticulocyte in vitro protein-synthesizing system. RBP was identified and separated from other translated products by immunoprecipitation with specific rabbit anti-rat RBP antiserum. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of the immunoprecipitate consistently revealed one major product which migrated more slowly than purified rat serum RBP. The protein (preRBP) had an approximate molecular weight of 24,000. When dog pancreas microsomal membranes were cotranslationally present, the newly synthesized preRBP was processed to a protein which migrated coincidentally with purified rat serum RBP, approximately 20,500 daltons. These results indicate that RBP is initially synthesized as a larger molecular weight precursor (preRBP) which is rapidly processed by the removal of a peptide of approximately 3,500 daltons to the size of the final RBP molecule that circulates in the plasma.