Biomedical subjects

R E McGehee

Publications and source records attributed to R E McGehee.

16 recordsLinked to original sources

PubMed2001

Antisense suppression of p107 inhibits 3T3-L1 adipocyte differentiation.

The murine 3T3-L1 preadipocyte cell line is well characterized for its capacity to undergo differentiation into adipocytes under appropriate hormonal stimulation. p107, a member of the retinoblastoma tumor suppressor gene family has been shown to be dramatically upregulated during the early requisite clonal expansion phase of 3T3-L1 adipogenesis; however, a functional consequence has yet to be described. A phosphorothioate antisense RNA approach was utilized to determine if inhibition of p107 expression would block or perturb adipocyte differentiation. A series of three phosphorothioate oligonucleotides in antisense orientation was generated, designated AS1, AS2, and AS3 along with a sense control oligonucleotide complementary to AS1 and added to postconfluent cells at a concentration of 20 and 50 microM throughout hormonally stimulated differentiation. Treatment of cells with either concentration of the sense, AS1, AS2, or 20 microM AS3 oligonucleotides had little effect on either Oil Red O lipid accumulation or induction of p107 protein levels. In contrast, treatment with 50 microM AS3 inhibited the increase in p107 protein levels and led to a complete block in differentiation as detected by Oil Red O lipid accumulation and inhibition of adipocyte-specific mRNA expression. In addition, treatment with AS3 led to a significant inhibition of cellular proliferation associated with clonal expansion. Combined, these results provide strong evidence supporting a functional role for p107 in 3T3-L1 adipocyte differentiation.

3T3 Cells↗

PubMed2001

Human milk contains detectable levels of immunoreactive leptin.

Leptin, the recently cloned product of the obese (ob) gene, is a 16 kDa-protein that acts as a circulating satiety factor. It also serves to regulate energy expenditure and may act as a counter regulatory hormone to insulin. Initially thought to be exclusively produced by mature adipocytes, its mRNA has now been identified in significant levels in the placenta as well as the fetus raising speculation regarding its importance as a growth factor. Given studies demonstrating that exclusively breast-fed infants are leaner due to decreased energy intakes than formula-fed infants, we hypothesized that the presence of leptin in human milk could participate in mediating the earlier satiety of those infants fed human milk. We undertook this initial study to qualitatively examine the presence of leptin in human milk utilizing an immunoblot approach. Random milk samples during the first 2 weeks of lactation were available for study from 4 mothers delivering at term. Milk samples were centrifuged, the aqueous layer removed, and the protein content quantitated. One-hundred micrograms of total protein were separated by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE), transferred to nitrocellulose, and immunoblotted with an antileptin antibody. As controls, recombinant human leptin alone and a sample of milk containing added leptin were similarly electrophoresed and immunoblotted. Labeled proteins were visualized by chemiluminescence. Significant amounts of leptin protein were identified in all milk samples examined. No difference in protein detection was identified in fresh milk vs. frozen milk, and little difference was apparent in foremilk samples vs. hindmilk samples. These preliminary data reveal the presence of leptin in term human milk and suggest that further studies to document bioactivity of milk-derived leptin are warranted.

Female↗

PubMed2000

Cyclic expression of class I alcohol dehydrogenase in male rats treated with ethanol.

Continuous infusion of ethanol-containing diets has been demonstrated to generate well-defined pulses in blood and urine ethanol concentrations that occur with a frequency of approximately 6 days. The present study aimed to determine if hepatic class I alcohol dehydrogenase was the cause of these cycles. Adult male rats were fed an ethanol-containing diet by continuous intragastric infusion. Hepatic ADH activity, class I ADH mRNA level and rate of class I ADH gene transcription fluctuated in a cyclic pattern that positively correlated with UECs, and inhibition of ADH with 4-methylpyrazole abolished the UEC pulses. These data demonstrate for the first time an ethanol-dependent regulation of rat hepatic class I ADH. The cyclic behavior of the ethanol levels correlates with changes in class I ADH expression and implies adaptability of the ethanol eliminating system to high concentrations of alcohol.

Alcohol Dehydrogenase↗

PubMed1999

Molecular regulation of adipocyte differentiation.

Significant advances have been made recently toward understanding the molecular events that regulate adipocyte differentiation. In vitro models of adipogenesis, such as the 3T3-L1 and F-442A preadipocyte cell lines have proven to be an invaluable resource in elucidating mechanisms of adipocyte differentiation. Subject to modulation by hormonal, dietary, and genetic influences, the differentiation program now appears to be distinctly controlled through the coordinate regulation of transcription factors that predominantly include members of the C/EBP and PPAR families. Increased understanding of these critical factors and how they are regulated will provide insights into adipose tissue development as well as treatment of obesity.

3T3 Cells↗

PubMed1998

TNFalpha disrupts mitotic clonal expansion and regulation of retinoblastoma proteins p130 and p107 during 3T3-L1 adipocyte differentiation.

The inhibitory effects of TNFalpha on adipocyte differentiation are well described, however, the mechanisms are poorly understood. Early during hormonally-induced 3T3-L1 preadipocyte differentiation there is a requisite mitotic clonal expansion phase that is associated with significant regulation in p130 and p107 protein levels, two members of the retinoblastoma protein family that regulate cell cycle events through interactions with the E2F transcription factors. This regulation occurs within the first 24 hours of differentiation (Day 1) and is characterized by a transient increase in p107 protein and mRNA levels as well as a transient decrease in p130 protein levels. Here we describe that TNFalpha disrupts the normal pattern of expression of both p130 and p107 proteins, leading to a complete block in mitotic clonal expansion. Interestingly, TNFalpha-treated cells enter S-phase as determined by 5-bromo-2'-deoxyuridine uptake experiments, but rather than completing cell cycle, they are stimulated to undergo apoptosis.

3T3 Cells↗

PubMed1998

Human milk and infant formula can induce in vitro adipocyte differentiation in murine 3T3-L1 preadipocytes.

The potential of infant diet to influence fat cell development has largely been examined in clinical studies with conflicting results. In this study, the direct effects of two standard infant formulas, Enfamil and Similac, as well as human milk were examined using a well characterized model of adipocyte differentiation, the 3T3-L1 murine preadipocyte cell line. After exposure to a hormonal regimen of insulin, dexamethasone, and 1-methyl-3-isobutylmethylxanthine, these cells undergo a mitotic expansion phase followed by terminal differentiation. On d 4 of hormonal exposure, greater than 95% of 3T3-L1 cells exhibit the morphologic and biochemical characteristics of mature adipocytes. In this study, cells were exposed to control medium, or control medium supplemented with either 10% Enfamil, 10% Similac, 10% human milk (skim or whole), or the standard hormonal regimen. Oil Red O-detectable lipid accumulation, immunocytochemical cell proliferation assays, and activated expression of adipocyte differentiation-specific mRNAs by Northern blot analysis were used to assess the effects of treatment on adipocyte differentiation. Results from each level of assessment revealed that both Enfamil and human milk were as effective as the standard hormonal regimen at stimulating adipocyte differentiation. In contrast, results from treatment with Similac or human skim milk were indistinguishable from control unstimulated cells. This study, demonstrating that Enfamil and human milk are capable of independently inducing in vitro adipocyte differentiation, suggests that diet during infancy could influence body fat development.

3T3 Cells↗

PubMed1997

Regulation and expression of retinoblastoma proteins p107 and p130 during 3T3-L1 adipocyte differentiation.

During 3T3-L1 adipocyte differentiation, growth-arrested, postconfluent preadipocytes are required to reenter the cell cycle and proceed through a mitotic clonal expansion phase prior to terminal differentiation. The retinoblastoma proteins (pRB, p107, and p130) are thought to be critical in controlling cell cycle progression by binding to and regulating the activity of the E2F transcription factors. We show here that p130/p107 protein levels, p107 mRNA levels, and E2F DNA binding complexes are regulated during 3T3-L1 adipogenesis. The predominant E2F binding complex in day 0 preadipocytes was p130-E2F with no detectable free E2F or p107. On Day 1, during mitotic clonal expansion, there was a distinct switch to free E2F and p107-E2F complexes associated with increased p107 mRNA and protein along with decreased p130 protein levels. Following differentiation, the day 0 pattern is reestablished. The switch is not just a consequence of reentry into the cell cycle, in that p107 protein levels are both detectable and unchanged in dividing, serum-restricted, or serum restimulated preconfluent cells. Interestingly, hormonal stimulation of 3T3-C2 cells, a related nondifferentiating cell line, also induces a mitotic clonal expansion phase that is associated with the p130:p107 switch in a pattern very similar to 3T3-L1 cells, suggesting the block in differentiation observed in 3T3-C2 cells occurs after clonal expansion. Combined, these findings suggest that the regulatory mechanisms of the p130:p107 switch are not specific to differentiation but may play a key role in regulating the mitotic clonal expansion necessary for adipocyte differentiation in 3T3-L1 cells.

3T3 Cells↗

PubMed1997

Regulation of the hepatic CYP 2E1 gene during chronic alcohol exposure: lack of an ethanol response element in the proximal 5'-flanking sequence.

Chronic exposure to ethanol is known to cause a dramatic increase in the level of CYP 2E1 apoprotein. More recently it has been demonstrated that under certain conditions the mRNA encoding cytochrome P450 2E1(CYP 2E1) is inducible; however, the mechanisms by which these increases occur are not well understood. In the current study, DNase I footprinting assays performed on the first kilobase of the CYP 2E1 5'-flanking sequences resulted in the identification of 13 sequence-specific protected regions using rat liver nuclear extracts isolated from either control or ethanol-treated animals. No differences were observed in the DNase I footprint patterns produced by the two different nuclear extracts. In addition, analysis by electrophoretic mobility shift assays (EMSA) revealed that with one exception, there were no differences in the level of binding complexes between the two extracts. However, EMSA analysis with an oligonucleotide to one footprint site (designated Site C) revealed that in nuclear extracts isolated from ethanol-treated animals there was a 2.9-fold increase in this binding complex when compared to control nuclear extracts. This site was previously shown to contain an HNF-1alpha binding site, and here we demonstrate that bacterially expressed HNF-1alpha in footprint assays bind Site C sequences and that HNF-1alpha transactivates the CYP 2E1 promoter in co-transfection experiments with HNF-1alpha expression plasmid and plasmids containing CYP 2E1 promoter sequences coupled to the chloramphenicol acetyl transferase gene. Furthermore, in contrast to the increase observed by EMSA in Site C binding, no increase was detected in the CYP 2E1 transcriptional rate supported by nuclear extracts from ethanol-treated animals over controls using in vitro transcription assays, suggesting that the increase by ethanol in CYP 2E1 transcription is not mediated through the HNF-1alpha site.

Animals↗

PubMed1996

Antisense oligonucleotides to differentiation-specific element binding protein (DSEB) mRNA inhibit adipocyte differentiation.

The Differentiation-Specific Element Binding Protein (DSEB) was identified by binding to a specific cis-acting DNA element (DSE) responsible for the irreversible continued expression of the angiotensinogen gene after differentiation of 3T3-L1 adipoblasts to adipocytes. It was also identified as the large subunit of the Replication Factor C complex. During 3T3-L1 adipoblast differentiation, DSEB is induced early and interacts with the DSE that is essential for the sustained transcriptional activation of the angiotensinogen gene. Here we describe loss of function studies in 3T3-L1 cells performed with antisense phosphorothioate oligonucleotides that hybridize to DSEB mRNA. Treatment with 15, 25, and 50 microM antisense DSEB resulted in a dose-dependent inhibition of differentiation-specific lipid accumulation after 3 days of hormonal stimulation. Similar treatment also markedly reduced differentiation-dependent expression of mRNAs encoding angiotensinogen and the fat-specific fatty acid binding protein, aP2. Further, 50 microM antisense DSEB treatment resulted in a significant approximately 50% inhibition of the cell proliferation that occurs early in 3T3-L1 adipogenesis. Control experiments using the DSEB sense oligonucleotide had no effect on hormonal-stimulated adipocyte differentiation. Combined, these results suggest that DSEB serves an important role during the proliferative phase of 3T3-L1 adipoblast differentiation.

3T3 Cells↗

PubMed1995

Differentiation-specific element binding protein (DSEB) binds to a defined element in the promoter of the angiotensinogen gene required for the irreversible induction of gene expression during differentiation of 3T3-L1 adipoblasts to adipocytes.

The differentiation-specific element (DSE) is a cis-acting transcriptional element located at nucleotide--1000 in the 5'-flanking promoter of the angiotensinogen gene. It is required for the irreversible and sustained increase in transcription of the angiotensinogen gene that occurs during differentiation of 3T3-L1 adipoblasts into adipocytes induced by a 3-day hormonal pulse. We report here the cloning of 3T3-L1 adipocyte cDNA encoding a 150 kilodalton protein designated Differentiation Specific Element Binding Protein (DSEB) that exhibits sequence-specific binding to a DSE oligonucleotide. Two DSEB mRNAs (3.6 and 4.2 kilobases) are observed in adipose, brain, kidney, testis, liver, and lung. Both DSEB mRNA and protein are induced during, and remain elevated after, 3T3-L1 cell adipogenesis. Analysis of adipoblasts by immunocytochemistry with an antiserum directed to bacterial expressed DSEB reveals that DSEB is localized to the nucleus and is induced during differentiation. DNA-binding assays show that binding is specific and exhibits high affinity and specificity for the DSE. Deletional analyses of bacterial expressed recombinant DSEB identifies a DNA-binding domain of 120 amino acids that contains two predicted helical regions. A sequence of 72 amino acids within the DNA-binding domain of DSEB is 60% identical to domains found in the sequences of several bacterial ligases. Further, DSEB is homologous to several proteins reported recently that are proposed to be a component(s) of the DNA replication-C complex raising the possibility that DSEB may be both a transcription factor and a DNA-replication factor.

3T3 Cells↗

PubMed1994

Characterization of cytochrome P450 2E1 induction in a rat hepatoma FGC-4 cell model by ethanol.

The hepatic microsomal ethanol-oxidizing system (MEOS) has been well characterized as an important pathway in ethanol metabolism. Cytochrome P450 2E1 (CYP 2E1), the principal component of MEOS, is ethanol inducible and has been implicated in hepatotoxicity associated with alcohol abuse and exposure to organic solvents. Results of chronic in vivo experiments have shown that ethanol induction of hepatic CYP 2E1 occurs by a two-step mechanism. The first step of induction is associated with low blood alcohol concentrations (BACs) and appears to be post-transcriptional, whereas high BACs observed in step-two induction are associated with increased CYP 2E1 gene transcription. The mechanisms underlying these induction steps are under intense investigation. Progress in this area has been limited due to lack of hepatic cell culture models that express CYP 2E1. We report here an in vitro tissue culture cell model, the FGC-4 hepatoma cell line, that exhibits basal levels of CYP 2E1 apoprotein that are inducible by ethanol treatment. Total cellular RNA and microsomal fractions were isolated from control or ethanol-treated confluent cells, and CYP 2E1 mRNA and apoprotein levels were characterized by northern blot or immunoblot analysis, respectively. Initial experiments on isolated microsomes revealed detectable levels of CYP 2E1 apoprotein in control cells that were induced 5-fold in cells treated with 100 mM ethanol for 24 hr. Concentration-response experiments demonstrated that the maximal 24-hr induction in CYP 2E1 apoprotein level was 5-fold and was attained at a concentration of 10 mM ethanol. Interestingly, while the steady-state mRNA levels encoding CYP 2E1 were detectable, they remained unchanged in identically treated cells. Furthermore, there was no observed increase in CYP 2E1 mRNA levels in an extended time course to 72 hr or at higher alcohol concentrations (up to 1500 mM), providing preliminary evidence that the induction is post-transcriptional. The time course of CYP 2E1 apoprotein induction by exposure to 100 mM ethanol demonstrated maximal induction at 8 hr. Measurement of CYP 2E1 apoprotein levels after removal of ethanol from pretreated cells demonstrated the half-life of the apoprotein to be 12.7 hr, in good agreement with previous reports using primary hepatocytes. The half-life of the induced protein after ethanol removal in the presence of cyclohexamide (10 micrograms/mL) was biphasic with a rapid 1.8 hr first phase followed by a slower 44.7 hr second phase.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1994

IDX-1: a new homeodomain transcription factor expressed in rat pancreatic islets and duodenum that transactivates the somatostatin gene.

We describe the cloning from a rat islet somatostatin-producing cell line of a 1.4 kb cDNA encoding a new homeoprotein, IDX-1 (islet/duodenum homeobox-1), with close sequence similarity to the Drosophila melanogaster homeobox protein Antennapedia (Antp) and the Xenopus laevis endoderm-specific homeoprotein XlHbox8. Analyses of IDX-1 mRNA and protein in rat tissues show that IDX-1 is expressed in pancreatic islets and ducts and in the duodenum. In electrophoretic mobility shift assays IDX-1 binds to three sites in the 5' flanking region of the rat somatostatin gene. In co-transfection experiments IDX-1 transactivates reporter constructs containing somatostatin promoter sequences, and mutation of the IDX-1 binding sites attenuates transactivation. Reverse transcription-polymerase chain reaction of islet RNA using degenerate amplimers for mRNAs encoding homeoproteins indicates that IDX-1 is the most abundant of 12 different Antp-like homeodomain mRNAs expressed in adult rat islets. The pattern of expression, relative abundance and transcriptional regulatory activity suggests that IDX-1 may be involved in the regulation of islet hormone genes and in cellular differentiation in the endocrine pancreas and the duodenum.

Amino Acid Sequence↗

PubMed1993

Differentiation-specific element: a cis-acting developmental switch required for the sustained transcriptional expression of the angiotensinogen gene during hormonal-induced differentiation of 3T3-L1 fibroblasts to adipocytes.

The gene encoding angiotensinogen, the glycoprotein precursor for the vasopressor angiotensin II, is under coordinate tissue-specific, developmental, and hormonal regulation. We show here that the irreversible, developmentally regulated increase in angiotensinogen gene expression during the hormonal (dexamethasone, insulin, and isobutylmethylxanthine) differentiation of fibroblast-like 3T3-L1 cells into adipocytes is mediated by a 14-base pair cis-acting element located at -1000 in the 5'-flanking region of the gene. The sequence of this differentiation-specific element (DSE) is similar to sites that bind the homeotic and pou class of transcription factors found in the promoters of other genes known to be regulated during differentiation. Furthermore, we show that there are several high affinity DSE-specific binding proteins present in preadipocyte nuclear extracts that are competed with known homeotic and pou transcription factor DNA binding sequences. Thus the DSE appears to serve as a developmental switch for the expression of the angiotensinogen gene during the differentiation of fibroblasts to adipocytes and may be a binding site for one or more of the pou-homeodomain class of transcription factors.

1-Methyl-3-isobutylxanthine↗

PubMed1992

Tumor necrosis factor-induced reversal of adipocytic phenotype of 3T3-L1 cells is preceded by a loss of nuclear CCAAT/enhancer binding protein (C/EBP).

Tumor necrosis factor (TNF)-treated 3T3-L1 adipocytes were used as a model for studying the effects of systemic inflammation on adipose tissue. Lipopolysaccharide-treated monocyte-conditioned medium or recombinant human TNF alpha induced morphological dedifferentiation of the adipocytes and led to loss of adipocyte specific gene expression. Gel shift, Southwestern and Western immunoblot analysis demonstrated that dedifferentiation was preceded by a decrease in the DNA binding activity and protein level of the transcription factor CCAAT/enhancer binding protein (C/EBP). Liver activating protein, a related protein that binds identical DNA sequences, increased during cytokine treatment. Both proteins activate specific enhancer elements located in the promoter region of many genes whose transcription is altered during systemic inflammation. Pulse-chase labeling followed by immunoprecipitation demonstrated that C/EBP is a rapidly turning over protein in adipocytes and that cytokine treatment led to a specific, time dependent decrease in its rate of synthesis. Because C/EBP binding sites have been shown to play an important role in regulating the expression of genes involved in adipocyte metabolism, we propose that the TNF-induced changes in the complement of transcription factors binding those sites may be important in the pathogenesis of inflammation-induced atrophy of adipose tissue.

3T3 Cells↗

PubMed1991

Alternative mRNAs encoding the alpha 1b-adrenergic receptor are expressed in a tissue-dependent manner in the Sprague-Dawley rat.

Probing total cellular and poly [A+] RNA isolated from various rat tissues with a full-length cDNA encoding the hamster alpha 1-adrenergic receptor results in detection of two transcripts, 3.3 kb and 2.7 kb, which probably both encode the alpha 1b-adrenergic receptor subtype. Both the 3.3 kb and 2.7 kb mRNAs were found to be associated with hepatic polysomes which suggests that these mRNA species are translated into protein. Using non-overlapping 5' and 3' cDNA probes, large sequence differences were not evident between the 3.3 kb and 2.7 kb mRNAs, although the 3'-probe hybridized to a 4.0 kb mRNA in addition to the two smaller transcripts in poly [A+] RNA isolated from renal cortex, but not other tissues. The relative amounts of the 3.3 kb and 2.7 kb mRNAs varied considerably among the five tissues studied. However, the ratio of the two transcripts remained relatively constant in the same tissue taken from animals at different developmental ages. Currently, the physiological significance of multiple alpha 1b-adrenergic receptor gene transcripts is unclear. However, our results suggest that alpha 1b-adrenergic receptor gene expression in the rat is under complex regulatory control that in part is tissue-dependent.

Animals↗

PubMed1990

Detection by northern analysis of alpha 1-adrenergic receptor gene transcripts in the rat.

In Northern blots of total cellular and poly(A+) RNA isolated from rat liver, renal cortex, spleen, and brain probed with a full-length cDNA encoding the hamster alpha 1-adrenergic receptor, hybridization was observed to two distinct mRNAs, at approximately 3.3 kb and approximately 2.7 kb. Only the approximately 2.7 kb mRNA species was visualized in Northern blots of total cellular and poly(A+) RNA isolated from cardiac ventricular muscle. From screening a rat heart cDNA library with the full-length hamster alpha 1-adrenergic receptor cDNA, a 632 base pair cDNA was isolated. Based upon its high degree of identity, 86% at the nucleotide level, with the hamster alpha 1-adrenergic receptor cDNA, this cDNA was considered to include the 3' end of the rat alpha 1-adrenergic receptor. When used as a probe in Northern blots of liver RNA, both the approximately 3.3 kb and approximately 2.7 kb mRNAs were visualized. Both mRNA species were expressed in fetal as well as adult liver, but steady-state levels of each gene transcript were approximately 3-fold higher in adult compared to fetal liver. Finally, results from Southern analysis of restriction enzyme fragments of genomic DNA suggest that the two gene transcripts may be products of a single gene.

Animals↗