Biomedical subjects
Publications and source records attributed to O A MacDougald.
As well as being the precursors of the triacylglycerols deposited as fat in adipose tissue, long-chain fatty acids are one class of agents that induce the differentiation of preadipocytes to adipocytes.
The effect of insulin on expression of CCAAT/enhancer binding protein (C/EBP) alpha, beta, and delta was investigated in fully-differentiated 3T3-L1 adipocytes. Treatment of adipocytes with insulin stimulated rapid dephosphorylation of C/EBP alpha, and repressed the expression of C/EBP alpha within 2-4 h, with > 90% suppression occurring at 24 h. While insulin induced expression of C/EBP beta and C/EBP delta within 1 h and caused a > 20-fold increase by 4 h, expression returned to nearly pretreatment levels by 24 h. The insulin concentration dependence of these effects was consistent with involvement of the insulin receptor. Gel shift analysis revealed that 6 h of insulin treatment decreased the binding of nuclear C/EBP alpha while increasing binding of nuclear C/EBP beta and C/EBP delta. The reciprocal effects of insulin on the steady-state levels of C/EBP transcription factors can be accounted for kinetically and quantitatively by changes in their mRNA levels, which can be accounted for by effects on gene transcription. The effects of insulin on adipocyte gene transcription (e.g. GLUT4) may be mediated, at least in part, by down-regulation of C/EBP alpha and/or its dephosphorylation.
Cell culture models (e.g. 3T3-L1 cells) have been developed for studying the process of adipocyte differentiation. Differentiation can be induced by adding insulin-like growth factor I, glucocorticoid, fatty acids, and an agent that increases intracellular cAMP level. The adipocyte differentiation program is regulated by transcriptional activators such as CCAAT/enhancer binding protein alpha (C/EBP alpha), peroxisomal proliferator activated receptor gamma 2 (PPAR gamma 2), fatty acid activated receptor (FAAR), and transcriptional repressors such as preadipocyte repressor element binding protein (PRE) and C/EBP undifferentiated protein (CUP). These transcription factors coordinate the expression of genes involved in creating and maintaining the adipocyte phenotype including the insulin-responsive glucose transporter (GLUT4), stearoyl CoA desaturase 1 (SCD1), and the fatty acid binding protein (422/aP2).
Glucocorticoid agonists, i.e. dexamethasone or triamcinolone acetonide, rapidly induce expression of CCAAT/enhancer-binding protein (C/EBP) delta and repress expression of C/EBP alpha in fully differentiated 3T3-L1 adipocytes. Within 30 min of glucocorticoid treatment, the cellular level of C/EBP delta rises dramatically, increasing > 100-fold within 6 h. Concurrently, the level of C/EBP alpha decreases, reaching a minimum within 4 h. The dexamethasone concentration dependence and steroid specificity of these responses suggest that both processes are mediated by the glucocorticoid receptor. The reciprocal effects of dexamethasone on the steady-state levels of C/EBP alpha and C/EBP delta can be accounted for kinetically and quantitatively by changes in their mRNA levels and by the transcription rates of their respective genes. The glucocorticoid-induced changes in expression of the C/EBP isoforms are correlated with the transcriptional activation of the SCD1 gene, an adipocyte gene known to be transactivated by C/EBP isoforms. Glucocorticoids also regulate expression of the C/EBP isoforms in vivo. Within 4 h of administration of dexamethasone or triamcinolone acetonide to adult rats, expression of C/EBP delta is induced in white adipose tissue while expression of C/EBP alpha is repressed. Like the response in 3T3-L1 adipocytes, the effects of dexamethasone on C/EBP alpha in white adipose tissue are rapid and transient.
The effect of exogenous fatty acids on expression of the stearoyl-CoA desaturase-1 (SCD1) gene was assessed both in vivo and ex vivo. Mice fed a fat-free diet or a diet containing a largely monounsaturated (18:1) fat, i.e., olive oil, expressed high levels of hepatic SCD1 mRNA. In contrast, in mice fed diets containing primarily polyunsaturated (18:2 and 18:3) fats, expression of the hepatic SCD1 message was markedly suppressed. Similar experiments with pure fatty acid esters showed that arachidonate (20:4) and linoleate (18:2) were far more potent in down-regulating expression of the hepatic SCD1 message than oleate (18:1), an end-product (as its CoA thioester) of the SCD1-catalyzed reaction. The reduction of hepatic SCD1 mRNA appears to be primarily due to inhibition of SCD1 gene transcription since polyunsaturated fatty acids caused a decrease in run-on transcription of the gene comparable to the decrease in message level. Consistent with the effects observed in vivo, unsaturated fatty acids suppressed the expression of SCD1 mRNA by rat hepatocytes cultured in serum-free medium. Suppression increased with degree of unsaturation with arachidonic (20:4) and eicosapentaenoic (20:5) acids, causing a > or = 90% reduction in the level of SCD1 message. Thus, the SCD1 gene, like the fatty acid synthase and S14 genes, undergoes coordinate transcriptional down-regulation in response to unsaturated fatty acids.
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Full-length (42 kDa) CCAAT/enhancer binding protein alpha (C/EBP alpha) (p42) has been implicated in the transcriptional activation of adipocyte genes including the 422(aP2) and C/EBP alpha genes during differentiation of 3T3-L1 preadipocytes. We have identified a 30-kDa isoform (p30) of C/EBP alpha that is expressed by 3T3-L1 adipocytes, mouse adipose tissue, and rat liver. In vitro translation of wild-type C/EBP alpha mRNA or transient transfection with a wild-type C/EBP alpha vector gave rise to similar levels of p42 and p30. Mutational analysis revealed that p30 is an alternative translation product initiated at the third in-frame methionine codon of the C/EBP alpha message. p30C/EBP alpha binds to the C/EBP sites within and activates reporter gene expression driven by the 422(aP2) and C/EBP alpha gene promoters. Although transfection of 3T3-L1 preadipocytes with a strong p30C/EBP alpha expression vector is insufficient to induce differentiation, this vector advances the differentiation program. Unlike p42C/EBP alpha, which inhibits cell proliferation, p30C/EBP alpha is not antimitotic. Thus, the N-terminal 12-kDa segment of full-length C/EBP alpha contains an amino acid sequence necessary for antimitotic activity. During differentiation of 3T3-L1 preadipocytes and during hepatocyte development, the cellular p42C/EBP alpha/p30C/EBP alpha ratio changes, raising the possibility of a regulatory role.
S14 gene transcription is induced by retinoic acid (RA) in cultured adipocytes, but not preadipocytes. Transfection of 3T3-L1 cells with S14-CAT fusion genes showed that adipocyte-specific RA responsive cis-acting elements are located between -1588 and -1381 bp upstream from the 5' end of the S14 gene. This region has enhancer-like properties. In contrast, an artificial RA response element conferred RA control to a CAT gene in both preadipocyte and adipocyte phenotypes indicating that the RA regulatory network is functional in both phenotypes. These studies show that RA receptors interact with adipocyte-specific transcription factors to control S14 gene expression.
Hepatic nuclear extracts support in vitro transcription from S14 and fatty acid synthase (FAS) promoters. Renal nuclear extracts support in vitro transcription from S14 promoter constructs at rates comparable to hepatic nuclear extracts, but lack the apparent positive and negative control observed with liver. In contrast, renal nuclear extracts do not support in vitro transcription from the FAS promoter. NF-1 or a related protein in both hepatic and renal extracts contributes to in vitro transcriptional activity from the S14 promoter, but not the FAS promoter. These studies indicate that different mechanisms regulate tissue-specific expression of S14 and FAS.
The structure of DNAase I hypersensitive site 1 (Hss-1), located adjacent to the 5' end of the rat liver S14 gene, is regulated by tissue-specific factors, and its formation correlates with the transcriptional activation of the S14 gene. We propose that tissue-specific trans-acting factors interacting with key cis-linked elements within this site function in the initiation of S14 gene transcription. To examine this hypothesis we used DNAase I footprint, gel shift and in vitro transcriptional analyses to identify cis-linked elements that function in the control of S14 gene transcription. Binding of rat liver nuclear proteins to the S14 promoter (from -8 to -464 bp) produced four DNAase I footprints (designated A-D). Gel shift studies showed that DNA-protein binding was tissue- and sequence-specific, differentially heat-sensitive, and abolished by proteinase K. The function of the four cis-acting elements was assessed by using an in vitro transcription initiation assay in which the S14 promoter was fused to a reporter gene (G-free cassette). Deletion studies showed that nuclear factors binding to regions A (-48 to -63 bp), B (-88 to -113 bp) and D (-286 to -310 bp) enhanced the rate of initiation of transcription, while proteins binding to region C (-227 to -244 bp) suppressed the rate of initiation of transcription. Based on oligonucleotide competition studies, we suggest that hepatic NF-1 (or a related protein) binding to the A region enhances the rate of initiation of S14 gene transcription. Since trans-acting factors interacting with regions B and D are found in liver but not in spleen or kidney, we suggest that the proteins interacting with these regions may be involved in the tissue-specific augmentation of S14 gene transcription.
Thirty-two gilts were used to evaluate the effects of increased dietary energy and CP during late gestation on mammary development. On d 75 of gestation, gilts were assigned randomly in a 2 x 2 factorial arrangement to adequate (5.76 Mcal ME/d) or increased (10.5 Mcal ME/d) energy and adequate (216 g CP/d) or increased (330 g CP/d) protein. On d 105 of gestation, gilts were slaughtered and total mastectomies were performed. Mammary tissue was separated into mammary parenchymal and mammary extraparenchymal stromal tissue and analyzed for DNA, RNA, protein and lipid. No interactions between dietary energy and protein level were detected (P greater than .20). When adjusted for number of mammary glands and maternal BW (weight of the sow less the weight of the fetuses), mammary parenchymal weight was 27% greater (P less than .03) in gilts fed adequate energy than in gilts fed increased energy, but mammary extraparenchymal stroma weight was unaffected by dietary energy level. Total mammary parenchymal DNA was 30% greater in gilts fed adequate energy than in gilts fed increased energy (P less than .03). Total mammary parenchymal RNA (P less than .02) and total mammary parenchymal protein (P less than .02) also were greater in gilts fed adequate energy than in gilts fed increased energy. Dietary protein level did not affect mammary variables measured, except that increased dietary protein tended to reduce mammary extraparenchymal stromal weight (P less than .09). Increased dietary protein between d 75 and d 105 of gestation did not benefit mammary development, but increased dietary energy was detrimental to development of mammary secretory tissue.
Two experiments were conducted to develop a vaginal epithelium bioassay for zearalenone (Z) and to determine whether immunization against Z would prevent Z mycotoxicosis. Eleven gilts were ovariectomized and allotted by weight to dietary doses of 50, 150 or 350 micrograms Z/kg BW daily for 3 d. All doses of Z increased height of the vaginal epithelium. Height of the vaginal epithelium in gilts fed 150 or 350 micrograms Z/kg BW increased more than that in gilts fed 50 micrograms Z/kg BW. Twenty-four gilts then were ovariectomized and allotted to be immunized or not immunized. A Z-bovine serum albumin conjugate was injected into gilts to achieve immunization. Ten weeks after initial immunization, antibodies to Z were detected after a 1:10(7) dilution at greater than .1 absorbance units using an enzyme-linked immunosorbent assay, and gilts were allotted by weight to diets with no Z or 150 micrograms Z/kg BW daily for 3 d. Immunization alone had no effect on height of vaginal epithelium, but after 3 and 10 d, immunized gilts fed Z had higher vaginal epithelium than did nonimmunized gilts fed Z. Immunized gilts excreted a larger percentage of ingested Z than nonimmunized gilts did. Therefore, immunizing gilts against Z potentiated both the estrogenic effects of Z and urinary excretion of Z equivalents.
A direct competitive enzyme-linked immunosorbent assay (ELISA) was modified for the determination of zearalenone and zearalenois in porcine urine. In the modified procedure, standard or unknown concentrations of zearalenone were added to all wells, followed by rapid addition of zearalenone-horseradish peroxidase conjugate, whereas in previous methods, zearalenone and zearalenone-horseradish peroxidase were mixed and then added to microtiter wells. The modification increased the number of urine samples that could be analyzed per assay. The linear portion of the modified ELISA standard curve covered the range of 10-500 ng zearalenone/mL. Average recovery of zearalenone from spiked urine was 91, 101, 95, 107, 136, 112, and 120% for 1, 10, 25, 50, 100, 250, and 500 ng zearalenone/mL urine, respectively. Mean within-assay coefficient of variation for each concentration of zearalenone in 5 standard curves was 5.95%. Between-assay coefficients of variation for concentrations of zearalenone equivalents in lower and upper regions of the standard curve were 10.9% (n = 18) and 8.8% (n = 20), respectively. Analysis of urine samples showed that a gilt excreted 5.3% of ingested zearalenone in the 8 h following ingestion, with an average excretion rate of about 100 micrograms zearalenone equivalents/h.