Search PubMed⌕ Search

Biomedical subjects

H S Sul

Publications and source records attributed to H S Sul.

At least 37 records · Page 2Linked to original sources
PubMed1997

Molecular mechanisms of adipocyte differentiation and inhibitory action of pref-1.

Commitment and differentiation of adipocytes is governed by transcription factors that are under the control of the combinatorial effects of hormonal and cell-cell and cell-matrix interaction. Established preadipocyte cell lines, such as 3T3-L1, 3T3-F442A, and Ob 17, have made it possible to examine the molecular details of the differentiation process. Differentiation is accompanied by dramatic increases in adipocyte genes, including adipocyte fatty acid-binding protein and lipid-metabolizing enzymes. Transcription factors PPAR gamma and C/EBP have been shown to transactivate some of the adipocyte-expressed genes. By integrating hormonal and metabolic cues, these nuclear factors may synergistically function in adipocyte lineage determination and differentiation. Adipocyte differentiation involves drastic cell shape alterations that are accompanied by changes in expression of cytoskeletal and extracellular matrix proteins, including decreases in actin and tubulin levels. Pref-1, an EGF-repeat containing transmembrane protein, is highly expressed in preadipocytes; this expression is totally abolished after differentiation to adipocytes. Pref-1 is inhibitory for adipocyte differentiation and processing of transmembrane pref-1 generates a biologically active soluble from corresponding to the ectodomain. Interaction of the EGF-repeats of pref-1 with an as yet unidentified receptor may mediate the inhibitory effects of pref-1 in adipocyte differentiation, thereby affecting nuclear events accompanying adipogenesis.

Adipocytes↗
PubMed1996

Characterization of Pref-1 and its inhibitory role in adipocyte differentiation.

Differentiation of preadipocytes to mature adipocytes is accompanied by marked increases in enzymes of lipid metabolism and cytoskeletal remodelling of fibroblastic preadipocytes to rounded lipid-filled mature adipocytes. Studies to date indicate this process is under the control of specific transcription factors, cell-cell and cell-environment interactions. Preadipocyte factor-1 (pref-1), a member of the EGF-like protein family, was identified during the differential screening of cDNAs expressed in differentiating preadipocytes. Multiple forms of the protein are present in 3T3-L1 preadipocytes with heterogeneity attributable both to alternate splicing and various post-translational modifications. Pref-1 is unique in that its expression is completely abolished during the maturation of preadipocytes to adipocytes. Moreover, blocking down-regulation via constitutive expression of pref-1 in preadipocytes inhibits their differentiation. The expression pattern of pref-1 in embryonic tissues and its presence in tumors indicate that in addition to its function in adipocyte differentiation, pref-1 may have a broader role in differentiation and development.

3T3 Cells↗
PubMed1995

Hormonal and nutritional control of the fatty acid synthase promoter in transgenic mice.

To study the molecular basis of tissue-specific and hormonally regulated expression of the fatty acid synthase (FAS) gene in vivo, we generated lines of transgenic mice carrying 2.1 kilobases of the 5'-flanking region (-2100 to +67) of the rat FAS gene fused to a chloramphenicol acetyltransferase (CAT) reporter gene. This reporter gene construct was strongly expressed in tissues that normally express high levels of FAS mRNA, which include liver and white adipose tissues. In contrast, CAT reporter activity was not detected in appreciable levels in lung, heart, kidney, and muscle tissues, which do not normally show significant levels of FAS activity. The relative levels of the CAT mRNA driven by the rat FAS promoter in various tissues of the transgenic animals approximated those of the endogenous mouse FAS mRNA. We also examined the hormonal and nutritional regulation of the FAS(2.1)-CAT reporter gene in transgenic mice. CAT activity was increased in both liver and white adipose tissue when fasted animals were refed a high carbohydrate, fat-free diet. These changes in CAT activity and CAT mRNA levels occurred in parallel to the changes in endogenous mouse FAS mRNA levels. On the other hand, fasting/refeeding did not change CAT activity appreciably in other tissues, such as muscle and brown adipose tissue. Administration of dibutyryl cAMP at the start of refeeding prevented an increase in CAT activity in liver. However, the cAMP effect was tissue-specific as cAMP treatment did not bring about change in CAT activity in adipose tissue. Next, to examine the effect of insulin, we made the transgenic mice insulin-deficient by streptozotocin treatment. Insulin treatment of the streptozotocin-diabetic mice increased both the CAT activity and CAT mRNA levels driven by the rat FAS promoter in liver and white adipose tissue. These changes in CAT expression by insulin paralleled those in endogenous FAS mRNA levels. Administration of glucocorticoids increased CAT activity in all tissues examined: liver, white and brown adipose tissues, lung, heart, and spleen. Overall, the first 2.1 kilobases of the 5'-flanking region of the rat FAS gene appear to contain sequence elements necessary to confer tissue-specific and hormonally regulated expression characteristic of the endogenous FAS gene.

Adipose Tissue↗
PubMed1995

Upstream stimulatory factors bind to insulin response sequence of the fatty acid synthase promoter. USF1 is regulated.

Fatty acid synthase (FAS) plays a central role in de novo lipogenesis in mammals. The concentration or activity of FAS in liver and adipose tissue changes dramatically when animals are subjected to nutritional and hormonal manipulations. We previously reported that due to changes in transcription, FAS synthesis declines and increases in an insulin-dependent manner during fasting and refeeding, respectively, and that insulin administration of streptozotocin-diabetic mice stimulates FAS transcription. We previously mapped the FAS insulin response sequence (IRS) to the proximal promoter region from position -71 to position -50, which contains an E-box DNA binding motif. Here, using competition gel shift assays and specific upstream stimulatory factor (USF) antibodies, we identified USF1 and USF2 as major components of complexes that bind to the FAS IRS. UV-cross-linking experiments further supported that USFs bind the FAS IRS. We also found that the amount of the 43-kDa USF1 was dramatically increased in liver of refed rats. In contrast, the amount of USF2 remained the same in liver of fasted or refed rats. Moreover, a 17-kDa protein in both fasted and refed rat liver was recognized by anti-USF1 antibodies, and this 17-kDa USF1-related protein was expressed in a manner opposite to that of the 43-kDa USF1, i.e. high in liver of fasted rats and decreased in liver of refed rats. These data suggest that the regulation of USF expression may play an important role in the regulation of FAS transcription.

Animals↗
PubMed1995

Purification and reconstitution of murine mitochondrial glycerol-3-phosphate acyltransferase. Functional expression in baculovirus-infected insect cells.

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial step in glycerolipid biosynthesis. We recently cloned a cDNA to a 6.8-kb mRNA, a message that can be induced dramatically by feeding a high-carbohydrate diet [Paulauskis & Sul (1988) J. Biol. Chem. 263, 7049-7054; Shin et al. (1991) J. Biol. Chem. 266, 23834-23839], and identified the open reading frame, p90, as mitochondrial GPAT [Yet et al. (1993) Biochemistry 32, 9486-9491]. To initiate characterization of mitochondrial GPAT, we purified and reconstituted the GPAT activity using phospholipids after expressing functional enzyme in Sf9 insect cells. Infection with recombinant virus containing p90 sequence resulted in high levels of GPAT expression in mitochondria, compared to noninfected cells or cells infected with the reverse orientation insertion baculovirus. There was a dramatic increase in N-ethylmaleimide-resistant mitochondrial GPAT activity. The GPAT protein was not detectable by Western blot in noninfected Sf9 cells or in cells infected with the GPAT sequence in the reverse orientation. However, in cells infected with GPAT in the correct orientation, there was a dramatic increase in the GPAT protein that was readily detectable by Coomassie staining both in total extracts and in the mitochondrial fraction. To ease the purification, we next expressed GPAT as a polyhistidine fusion protein in insect cells. The polyhistidine tag did not interfere with targeting to mitochondria or with the catalytic activity of GPAT.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Characterization of the murine mitochondrial glycerol-3-phosphate acyltransferase promoter.

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the acylation of sn-glycerol 3-phosphate to form 1-acyl-sn-glycerol 3-phosphate, a committed step in triacylglycerol and phospholipid biosynthesis. We have previously reported the cDNA cloning and transcriptional regulation of the murine mitochondrial GPAT (mGPAT). We now report the cloning of the 5'-flanking region of the murine mitochondrial GPAT. The transcription start site was identified by primer extension and RNase protection assays. A TATA box-like motif (TTATTAT) was located between -34 and -29 and a reverse CCAAT box (ATTGG) was located between -78 and -74, relative to the transcription start site. To begin studying mechanisms underlying transcriptional regulation of the mGPAT gene, chimeric luciferase (LUC) plasmids containing serial deletions, from -1447 to -38, of the 5'-flanking region of the murine mGPAT gene were prepared and transfected into 3T3-L1 cells. The fusion construct -1447 GPAT.LUC showed high promoter activity and deletions to -1353, -747, -322, and -86 did not markedly change the promoter activity. With all constructs, luciferase activity was 2-fold higher when plasmids were transfected into 3T3-L1 adipocytes. However, deletion of sequences between -86 and -55 resulted in a 9-fold decrease in LUC activity in both preadipocytes and adipocytes. Deletion of sequences between -55 and -38 did not alter promoter activity. DNase I footprint analysis revealed a protected region between -95 and -65 which included the putative CTF/NF1 binding site. Electrophoretic mobility shift assays demonstrated a single protein-DNA complex formation. Oligonucleotides synthesized according to the CTF/NF1 consensus sequence or the adenovirus NF-1 site showed a different and more complex pattern of protein-DNA interaction and were not able to compete away the mGPAT promoter-protein complex, indicating that a distinct protein was bound to -86/-55, a region important for the basal promoter activity in 3T3-L1 cells. Luciferase activity was increased 2.8- and 8-fold when adipocytes stably transfected with -322 GPAT.LUC were treated with 5 and 25 mM glucose, respectively, in the presence of 10 nM insulin. These results indicate that carbohydrate-responsive sequences are located within -322 base pairs of the mGPAT promoter.

3T3 Cells↗
PubMed1994

Structural characterization and alternate splicing of the gene encoding the preadipocyte EGF-like protein pref-1.

Preadipocyte factor 1 (pref-1), a member of the EGF-like protein family, is a transmembrane protein with six tandem EGF-like repeats in the putative extracellular domain. Expression of pref-1 is abolished during the in vitro differentiation of 3T3-L1 preadipocytes to adipocytes, and constitutive expression of pref-1 in preadipocytes inhibits their differentiation [Smas, C.M., & Sul, H.S. (1993) Cell 73, 725-734]. In the present studies, we have isolated and characterized genomic clones for pref-1 and have identified multiple pref-1 transcripts generated by alternate splicing. The pref-1 gene consists of five exons and four introns spanning approximately 7.3 kb. By primer extension analysis, the transcription start site was determined to be 169 bp upstream from the translation initiation codon. We have identified functional promoter sequences by transient transfection using a 2.1 kb fragment of the pref-1 5' flanking region linked to a luciferase gene; the pref-1-luciferase fusion gene construct gave 20-fold higher promoter activity as compared to the promoterless vector. Analysis of exon-intron junctions reveals that unlike the majority of the mammalian EGF-like genes, EGF-like repeats of pref-1 are not encoded by discrete exons. Through RT-PCR and the isolation and analysis of multiple pref-1 cDNA clones, we have identified, in addition to full-length pref-1, five alternately spliced forms with various in-frame deletions of all or a part of the sixth EGF-like repeat, juxta-membrane, and predicted transmembrane domains.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗
PubMed1994

Localization of sequences for the basal and insulin-like growth factor-I inducible activity of the fatty acid synthase promoter in 3T3-L1 fibroblasts.

Fatty acid synthase (FAS) plays a central role in fatty acid synthesis and its expression is under nutritional and hormonal control. We have investigated insulin-like growth factor-I (IGF-I) regulation of FAS by transfecting into 3T3-L1 fibroblasts chimeric genes comprising the 5'-flanking region of the FAS gene linked to a luciferase (LUC) reporter gene. First, the basal promoter activity of the 5' serial deletions from nucleotides -318 to -19 of the FAS gene were compared. Deletions of the promoter sequences from -136 to -19 resulted in a step-wise decrease in the promoter activity, with the -67 LUC and -19 LUC plasmids retaining 40% and 16% of the luciferase activity of -136 LUC. Regulatory sequences important for the FAS basal promoter activity in 3T3-L1 fibroblasts are, therefore, located within the -136 to -19 region. Treatment with 10 mM IGF-I also increased luciferase activity 1.8 +/- 0.2-, 1.8 +/- 0.3- and 2.5 +/- 0.1-fold in 3T3-L1 fibroblasts transiently transfected with -136 LUC, -110 LUC and -67 LUC plasmids, respectively. Deletion of sequences from -67 to -19 resulted in the loss of responsiveness to IGF-I. Physiological doses of insulin (10 nM), however, did not increase luciferase activity in 3T3-L1 fibroblasts transfected with any of the above plasmids. Only upon treatment with pharmacological doses of insulin (1 microM), probably through IGF-I receptor, did luciferase activity increase 4.3 +/- 0.4-, 3.2 +/- 0.4- and 3.5 +/- 0.5-fold when transfected with -136 LUC, -110 LUC and -67 LUC plasmids, respectively; there was no increase with -19 LUC. The half-maximal effect of IGF-I on FAS promoter activity was observed at 3 nM and a maximal effect was reached at 10 nM. These results indicate that the increased promoter activities observed are probably mediated through the IGF-I receptor. Furthermore, sequences responsible for IGF-I regulation of the FAS gene are located within the proximal promoter between nucleotides -67 and -19 of the FAS gene.

1-Methyl-3-isobutylxanthine↗
PubMed1994

Identification of an insulin response element in the fatty acid synthase promoter.

We have previously reported that insulin increases fatty acid synthase (FAS) gene transcription, and that sequences responsible for positive regulation are located within the first 332 base pairs of the FAS promoter. To define minimal sequences required for insulin regulation within this region, chimeric constructs containing serial 5' deletions starting at -318 and extending through position +67 of the rat FAS gene ligated to the luciferase reporter gene were transfected into 3T3-L1 adipocytes. Insulin treatment at 10 nM increased luciferase activity 2-3-fold in 3T3-L1 adipocytes transfected with constructs containing progressive deletions from -318 to -67. This stimulation of the FAS promoter activity by insulin was dose-dependent. However, no effect of insulin was observed when fusion constructs containing FAS promoter sequences spanning from -25 or from -19 to +67 were transfected into adipocytes. These results suggest that the insulin response sequences of the FAS gene may be located in the region from -67 to -25. DNase I footprinting using liver nuclear extracts revealed a protected region spanning -71 and -50 in addition to a region near the putative TATA box. Gel mobility shift assays using the sequence from -71 to -50 as a probe revealed nuclear factor(s) from mouse liver and 3T3-L1 adipocytes that specifically complexed with this sequence. Mutational analysis of this region showed that sequences between -68 and -60 are essential for recognition and interaction with a trans-acting factor(s). Moreover, when three tandem repeats of the sequences spanning -68 to -52 were linked to the SV40 promoter and used for transfection, luciferase activity increased 3.6-fold in response to insulin treatment. Thus, we have identified novel cis-acting DNA sequences responsible for insulin regulation of the FAS gene, which interact with nuclear protein(s) from liver and adipocytes and which are found to share limited homology to insulin response sequences present in other genes.

3T3 Cells↗
PubMed1994

Control of the murine phosphofructokinase-A gene during muscle differentiation.

The muscle-specific isoform of phosphofructokinase (PFK-A) is induced during muscle development. To understand expression of PFK at the molecular level, transcription of the mouse PFK-A gene was examined during C2 myoblast differentiation to myotubes. PFK-A gene transcription increased 5-7-fold during differentiation in vitro. To identify cis-acting elements which direct muscle-specific transcription of the PFK-A gene, its 5'-flanking region and first exon were cloned and characterized. S1 nuclease protection and primer extension assays showed four sites of transcription initiation at 106, 105, 88, and 87 bp upstream of the translation initiation codon. Stable transfection of fusion genes linking -1900 to +99 of PFK-A 5'-flanking sequence to chloramphenicol acetyltransferase coding sequences into myogenic C2 cells did not confer muscle-specific expression. However, larger fragments of PFK-A 5'-flanking region (-5800 to +99) showed muscle-specific expression by transient transfection assay. The sequences directing muscle-specific transcription were further defined by linking various PFK-A upstream fragments to the luciferase gene under the control of the PFK-A proximal promoter, -335 to +99 bp. We found DNA sequence responsible for muscle-specific expression of the PFK-A gene between -4800 and -3900 bp.

Animals↗
PubMed1994

Structure, distribution, and functional expression of the phosphofructokinase C isozyme.

To elucidate the structure, tissue-specific expression, and allosteric properties of phosphofructokinase-C (PFK-C), we cloned the cDNA for PFK-C from a rat hypothalamic cDNA library. The cDNA is 2643 base pairs long and encodes a protein of 765 amino acids. The deduced amino acid sequence is highly homologous to PFK-M (muscle) and PFK-L (liver), 69 and 65% amino acid identity, respectively, especially at substrate binding and catalytic sites, while the allosteric binding sites are less conserved. Tissue-specific expression of PFK-C was investigated by Northern blot analysis. PFK-C mRNA was detected in several brain regions and the anterior pituitary but not in liver, skeletal muscle, or several other tissues. In situ hybridization showed that PFK-C is expressed at a higher level in higher brain regions such as the cortex, compared with the midbrain and basal ganglia, while PFK-L is expressed at approximately equal levels throughout the brain. Expression plasmids containing PFK-C and PFK-L coding sequences were constructed and expressed by transient transfection into CMT cells. Expression of transfected PFKs was demonstrated by PFK enzymatic activity and by Western blotting with anti-rat brain and liver PFK antisera. Allosteric regulatory properties of PFK-C and PFK-L expressed in CMT cells were compared. Fructose 2,6-bisphosphate, a potent activator of PFK, decreased the Km of PFK-C for fructose 6-phosphate from 200 to 60 microM while decreasing that of PFK-L from 300 to 55 microM. The properties of PFK-C and PFK-L expressed in CMT cells clearly demonstrate the allosteric differences between the different PFK isozymes.

Allosteric Regulation↗
PubMed1993

Expression and identification of p90 as the murine mitochondrial glycerol-3-phosphate acyltransferase.

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial and committed step in glycerolipid biosynthesis. Mitochondrial GPAT, unlike the microsomal isozyme, prefers saturated fatty acids as a substrate. We have recently reported cloning of a cDNA to an unidentified 6.8-kb mRNA by a differential hybridization. The mRNA contains an open reading frame of 827 amino acids (p90) with 30% sequence homology in a 300 amino acid stretch to Escherichia coli GPAT. The 6.8-kb mRNA was induced dramatically when fasted mice were refed a high-carbohydrate diet. Here, we have expressed the open reading frame as trpE fusion proteins and used them to generate antibodies. The antibodies recognized a polypeptide of 90 kDa (p90) when the 6.8-kb cDNA sequence was used for in vitro transcription and translation. By Western blot analysis using these antibodies, we detected p90 in mitochondrial fractions of liver, and the p90 level was increased by refeeding. The increase in the p90 level correlated with the increase in mitochondrial GPAT activity. Moreover, p90 was not detectable in 3T3-L1 preadipocytes but markedly increased during adipose conversion. This increase was consistent with the 11-fold increase we observed in N-ethylmaleimide (NEM)-resistant mitochondrial GPAT activity during adipocyte differentiation. In addition, we have expressed p90 in CHO cells by stable transfection. The transfected genes in both correct and reverse orientations produced distinct 3.9-kb transcripts owing to the truncation of a part of the noncoding regions of the endogenous 6.8-kb mRNA before insertion into the pMSXND vector. The transfected CHO cells were treated with 2-aminopurine, an agent that increases expression of exogenous genes.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗
PubMed1993

Regulation of fatty acid synthase gene transcription. Sequences that confer a positive insulin effect and differentiation-dependent expression in 3T3-L1 preadipocytes are present in the 332 bp promoter.

We have previously reported induction of fatty acid synthase (FAS) gene expression by insulin and adipocyte differentiation in 3T3-L1 cells. In order to identify sequences responsible for insulin regulation of the FAS gene, chimaeric constructs containing serial deletions of the 5'-flanking region of the rat FAS gene ligated to the chloramphenicol acetyltransferase (CAT) reporter gene were prepared and transfected into 3T3-L1 cells. Plasmids containing 2100 (-2100CAT), 1400 (-1400CAT), 1009 (-1009CAT) and 332 (-332CAT) bp of FAS 5' flanking sequences exhibited comparable basal CAT activities in 3T3-L1 preadipocytes. This activity was 3-fold higher when these constructs were transiently transfected into 3T3-L1 adipocytes. Stably transfected 3T3-L1 cells also exhibited a 3-fold increase in CAT activity upon adipocyte differentiation, indicating that sequences required for the differentiation-dependent increase in FAS expression are located within the 332 bp promoter. Treatment with 10 nM insulin increased CAT activity by 2.1 +/- 0.2-, 2.6 +/- 0.1-, 2.0 +/- 0.2- and 1.7 +/- 0.2-fold respectively in 3T3-L1 adipocytes transiently transfected with -2100CAT, -1400CAT, -1009CAT and -332CAT plasmids. CAT activity was increased by 3.0 +/- 0.3- and 3.5 +/- 0.6-fold respectively by insulin treatment in adipocytes stably transfected with -2100CAT and -1009CAT plasmids. When insulin-responsive H4IIE hepatoma cells were transiently transfected with -2100CAT, -1400CAT, -1009CAT and -332CAT plasmids and then treated with 10 nM insulin, CAT activity increased by 3.1-, 3.1 +/- 0.8-, 3.0 +/- 0.7- and 2.3 +/- 0.5-fold respectively in serum-free media, and by 2.6 +/- 0.4-, 3.3 +/- 0.9-, 3.1 +/- 0.4- and 2.9 +/- 0.6-fold respectively in the presence of 0.5% serum. These results indicate that sequences responsible for insulin regulation of FAS gene are also located within 332 bp of the transcription start site.

3T3 Cells↗
PubMed1993

Pref-1, a protein containing EGF-like repeats, inhibits adipocyte differentiation.

With the aim of identifying novel regulators of adipocyte differentiation, we have cloned and characterized preadipocyte factor 1 (pref-1), a novel member of the epidermal growth factor (EGF)-like family of proteins. Pref-1 is synthesized as a transmembrane protein with six tandem EGF-like repeats. In preadipocytes, multiple discrete forms of pref-1 protein of 45-60 kd are present, owing in part to N-linked glycosylation. While pref-1 mRNA is abundant in preadipocytes, its expression is completely abolished during differentiation of 3T3-L1 preadipocytes to adipocytes. Moreover, constitutive expression of pref-1 in preadipocytes, which in effect blocks its down-regulation, drastically inhibits adipose differentiation. This indicates that pref-1 functions as a negative regulator of adipocyte differentiation, possibly in a manner analogous to EGF-like proteins that govern cell fate decisions in invertebrates.

Adipose Tissue↗
PubMed1991

Transcriptional regulation of p90 with sequence homology to Escherichia coli glycerol-3-phosphate acyltransferase.

We have previously isolated cDNA clones for several mRNAs that are dramatically increased in livers of fasted mice refed a high carbohydrate diet. We report here the sequence and regulation of one such mRNA; the 6.8-kilobase mRNA has an open reading frame of 2481 nucleotides, and the coded protein contains 827 amino acid residues (Mr of 90,000) with a 30% identity and an additional 42% similarity in an approximately 300-amino acid stretch to Escherichia coli glycerol-3-phosphate acyltransferase. The p90 mRNA is highly expressed in liver and in adipose tissue. When previously fasted mice were refed a high carbohydrate, fat-free diet, the liver mRNA level for p90 was increased about 20-fold at 8 h. Administration of dibutyryl cAMP at the time of refeeding prevented the increase in the p90 mRNA by 70%. In addition, there was no increase in the p90 mRNA level when previously starved streptozotocin-diabetic mice were refed. In diabetic animals, the p90 mRNA level increased by 2-fold 1 h after insulin injection and reached a maximum of 19-fold after 6 h. The increase in transcription rate of the p90 gene preceded that of steady state mRNA level caused by fasting/refeeding, and cAMP abolished the increase in transcription. Transcription of the p90 gene was not detectable in either fasted or refed streptozotocin-diabetic mice, but increased 4-fold 30 min after insulin administration and further increased up to 8-fold at 2 h. On-going protein synthesis was necessary for this increase.

Amino Acid Sequence↗
PubMed1991

Regulation of expression of the fatty acid synthase gene in 3T3-L1 cells by differentiation and triiodothyronine.

We have previously reported that fatty acid synthase mRNA levels increase 10-15-fold during the differentiation of 3T3-L1 cells to adipocytes, correlating well with the increase in the relative rate of synthesis for this enzyme. Here we show by transcription run-on assays that fatty acid synthase is highly transcribed in both preadipocytes and adipocytes. Furthermore, the transcription rate of the fatty acid synthase gene increased only 1.5-fold during the adipose conversion, whereas the apparent mRNA half-life increased from 2.5 h in preadipocytes to approximately 20 h in adipocytes. These results indicate that the increase in mRNA level during adipose conversion is not only due to the transcriptional activation of this gene but reflects the post-transcriptional stabilization of the message in adipocytes compared to preadipocytes. As thyroid hormone has been reported to increase lipogenic enzyme activities including fatty acid synthase in adipose tissue and in differentiating adipocytes in vitro, we used fully differentiated 3T3-L1 adipocytes to study T3 regulation of mammalian fatty acid synthase expression. We measured the effect of T3 on the relative rate of protein synthesis, mRNA content, and transcription rate of this gene. When mature adipocytes were treated with 10 nM T3, the relative rate of synthesis of fatty acid synthase increased 1.9-fold at 6 h, and it reached a maximum of 2.9-fold at 12 h. In addition, Northern blot analysis showed that T3 increased the steady-state mRNA level for fatty acid synthase by 2.4-fold at 12 h and 4.5-fold at 24 h. Furthermore, run-on transcription analysis with isolated nuclei from cells treated with T3 showed that the transcription rate of the fatty acid synthase gene increased 4.1-fold after 6 h of T3 treatment and remained at the stimulated level for 24 h. These results demonstrate that the increase in transcription of the fatty acid synthase gene preceded that of the steady-state mRNA level, indicating that T3 regulates expression of fatty acid synthase primarily by modulating the transcription rate of the gene. In conclusion, while the differentiation-dependent increase in fatty acid synthase is mediated by both transcriptional and posttranscriptional processes, T3 regulation is primarily at the transcriptional level.

3T3 Cells↗