scholarly journals Induction of transcripts derived from promoter III of the acetyl-CoA carboxylase-α gene in mammary gland is associated with recruitment of SREBP-1 to a region of the proximal promoter defined by a DNase I hypersensitive site

2003 ◽  
Vol 375 (2) ◽  
pp. 489-501 ◽  
Author(s):  
Michael C. BARBER ◽  
Amanda J. VALLANCE ◽  
Helen T. KENNEDY ◽  
Maureen T. TRAVERS
Keyword(s):  
Mammary Gland ◽  
De Novo ◽  
Dnase I ◽  
Proximal Region ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Proximal Site

ACC-α (acetyl-CoA carboxylase-α), a key regulator of fatty-acid metabolism, is encoded by mRNAs transcribed from three promoters, PI, PII and PIII, in the ovine genome. Enhanced expression of transcripts encoded by PIII in mammary gland during lactation is associated with alterations in chromatin structure that result in the detection of two DNase I hypersensitive sites, upstream of the start site. The most proximal site, located between −190 and −10, is characterized by the presence of an inverted-CCAAT box, C2 at −167, and E-boxes, E1 and E2, at −151 and −46. Deletion of these motifs, which bind nuclear factor-Y and upstream stimulatory factors respectively in gel-shift assays, attenuates the activity of luciferase reporter constructs in transfected cells. Chromatin immunoprecipitation demonstrated that these transcription factors were associated with PIII in vivo in both lactating and non-lactating mammary tissues. The basic helix–loop–helix-leucine zipper transcription factor, SREBP-1 (sterol-regulated-element-binding protein-1), transactivated PIII reporter constructs in transfected HC11 mammary cells, and this was dependent on the presence of E1, but not on C2 or E2. SREBP-1 was only associated with PIII in chromatin from lactating animals, which was coincident with a 4-fold increase in the precursor (125 kDa) form of SREBP-1 in microsomes and the appearance of the mature form (68 kDa) in the nucleus. SREBP-1 motifs are also present in the proximal region of PII, which is also induced in lactation. This indicates that SREBP-1 is a major developmental regulator of the programme of lipid synthesis de novo in the lactating mammary gland.

scholarly journals STAT5 binding contributes to lactational stimulation of promoter III expressing the bovine acetyl-CoA carboxylase alpha-encoding gene in the mammary gland

2002 ◽  
Vol 29 (1) ◽  
pp. 73-88 ◽  
Author(s):  
J Mao ◽  
AJ Molenaar ◽  
TT Wheeler ◽  
HM Seyfert
Keyword(s):  
Mammary Gland ◽  
De Novo ◽  
Point Mutations ◽  
Luciferase Reporter ◽  
Acetyl Coa Carboxylase ◽  
Luciferase Reporter Gene ◽  
Acetyl Coa ◽  
Rnase Protection ◽  
Lactating Mammary Gland ◽  
Encoding Gene

Activity of acetyl-CoA carboxylase (ACC)-alpha is rate limiting for de novo synthesis of fatty acids. The encoding gene is expressed by three different promoters. We characterized promoter III (PIII) from cow, previously only known from sheep. Quantitation of transcripts by RNAse protection assays and real time PCR revealed that PIII is primarily expressed and strongly induced ( approximately 28-fold) in the lactating mammary gland. PIII transcripts are expressed in mammary epithelial cells (MEC) as shown by in situ hybridization. A 2999 bp segment of the PIII promoter conferred prolactin and dexamethasone inducibility to a luciferase reporter gene in stably transfected mouse MEC cells. Lactogenic induction was abolished if a unique signal transducer and activator of transcription (STAT)-binding site at position -797 was inactivated by two point mutations. An oligonucleotide probe harboring this STAT-site specifically bound nuclear proteins from the lactating mammary gland. Binding was abolished by those two point mutations and super-shift analyses showed that STAT5A factors are present in this complex. Hence, prolactin, acting through STAT5, contributes to the activation of ACC expression in the milk producing cells of the lactating mammary gland. We discuss that STAT5 might be important in determining the milk composition by coordinating fatty acid and protein synthesis during lactation.

scholarly journals Regulation of acetyl-CoA carboxylase in rat mammary gland. Effects of starvation and of insulin and prolactin deficiency on the fraction of the enzyme in the active form in vivo

1982 ◽  
Vol 204 (1) ◽  
pp. 273-280 ◽  
Author(s):  
Elizabeth M. McNeillie ◽  
Victor A. Zammit
Keyword(s):  
Mammary Gland ◽  
Enzyme Activities ◽  
Enzyme Concentration ◽  
Acetyl Coa Carboxylase ◽  
Initial Activity ◽  
Acetyl Coa ◽  
Activity Ratios ◽  
Rat Mammary Gland ◽  
Rate Limiting

The ‘initial’ (I), endogenous phosphatase-activated (A) and citrate-activated (C) activities of acetyl-CoA carboxylase were measured in mammary-gland extracts of pregnant and lactating rats. There was a 10-fold increase in the A and C enzyme activities in the transition from early to peak lactation [cf. data of Mackall & Lane (1977) Biochem. J.162, 635–642], but there was no significant increase in the ratio of the initial activity to the A and C activities of the enzyme. Starvation (24h) or short-term (3h) streptozotocin-induced diabetes both resulted in a 40% decrease in I/A and I/C activity ratios. In starvation this was accompanied by a decrease in the absolute values of the A and C activities such that the initial activity in mammary glands of starved animals was 45% that in glands from fed animals. Insulin treatment of starved or diabetic animals 60min before killing increased the I activity without affecting the A or C enzyme activities. Removal of the pups for 24h from animals in peak lactation (weaning) resulted in a marked but similar decrease in all three activities such that, although the initial activity was only 10% of that in suckled animals, the I/A and I/C activity ratios remained high and unaltered. Inhibition of prolactin secretion by injection of 2-bromo-α-ergocryptine gave qualitatively similar results to those during weaning. Simultaneous administration of ovine prolactin completely prevented the effects of bromoergocryptine. It is suggested that the initial activity of acetyl-CoA carboxylase in rat mammary gland is regulated by at least two parallel mechanisms: (i) an acute regulation of the proportion of the enzyme in the active state and (ii) a longer-term modulation of enzyme concentration in the gland. Insulin appeared to mediate its acute effects through mechanism (i), whereas prolactin had longer-term effects on enzyme concentration in the gland. A comparison of initial enzyme activities (I) obtained in the present study with rates of lipogenesis measured in vivo [Agius & Williamson (1980) Biochem. J.192, 361–364; Munday & Williamson (1981) Biochem. J.196, 831–837] gave good agreement between the two sets of data for all conditions studied except for 24h-starved and streptozotocin-diabetic animals. It is suggested that acetyl-CoA carboxylase activity is rate-limiting for lipogenesis in the mammary gland in normal, fed, suckled or weaned animals but that in starved and short-term diabetic animals changes in the activity of the enzyme by covalent modification alone may not be sufficient to maintain the enzyme in its rate-limiting role.

scholarly journals Identification of a novel distal enhancer in human adiponectin gene

2008 ◽  
Vol 200 (1) ◽  
pp. 107-116 ◽  
Author(s):  
Katsumori Segawa ◽  
Morihiro Matsuda ◽  
Atsunori Fukuhara ◽  
Kentaro Morita ◽  
Yosuke Okuno ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Promoter Region ◽  
Luciferase Activity ◽  
Proximal Region ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Adiponectin Gene ◽  
Distal Enhancer

Adiponectin is exclusively expressed in adipose tissue and secreted from adipocytes, and shows anti-diabetic and anti-atherogenic properties. However, the precise transcriptional mechanism of adiponectin remains elusive. In this study, the 5′ flanking promoter region of human adiponectin gene was analyzed using UCSC genome browser, and a 10 390-bp fragment, containing an evolutionally conserved region among species, was investigated. The luciferase reporter assay using this fragment identified a novel distal enhancer of human adiponectin gene. Promoter constructs with the distal enhancer exhibited high promoter activities in 3T3-L1 mature adipocytes. However, no such activity was observed in other types of cell lines. The distal enhancer is highly conserved, and contains two completely conserved CCAAT boxes. In 3T3-L1 mature adipocytes, deletion or each point mutation of these CCAAT boxes markedly reduced luciferase activity driven by adiponectin promoter. Knockdown of CCAAT/enhancer-binding protein α (CEBPA; also known as C/EBPα) using small interfering RNA diminished adiponectin mRNA expression and luciferase activity driven by adiponectin promoter with the distal enhancer. However, adiponectin promoter with each mutation of two CCAAT boxes in the distal enhancer did not respond to knockdown of CEBPA expression. Furthermore, CEBPA bound to the distal enhancer both in vitro and in vivo. We also identified a proximal promoter region responsible for transcriptional activation by the distal enhancer in human adiponectin gene. Our results indicate that CEBPA plays a pivotal role in the transcription of human adiponectin gene via the distal enhancer and proximal region in its promoter.

Promoter I of the ovine acetyl-CoA carboxylase-α gene: an E-box motif at −114 in the proximal promoter binds upstream stimulatory factor (USF)-1 and USF-2 and acts as an insulin-response sequence in differentiating adipocytes

2001 ◽  
Vol 359 (2) ◽  
pp. 273-284 ◽  
Author(s):  
Maureen T. TRAVERS ◽  
Amanda J. VALLANCE ◽  
Helen T. GOURLAY ◽  
Clare A. GILL ◽  
Izabella KLEIN ◽  
...  
Keyword(s):  
Hepg2 Cells ◽  
Binding Protein ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Acetyl Coa Carboxylase ◽  
Upstream Stimulatory Factor ◽  
Acetyl Coa ◽  
Rnase Protection ◽  
E Box ◽  
Stimulatory Factor

Acetyl-CoA carboxylase-α (ACC-α) plays a central role in co-ordinating de novo fatty acid synthesis in animal tissues. We have characterized the regulatory region of the ovine ACC-α gene. Three promoters, PI, PII and PIII, are dispersed throughout 50kb of genomic DNA. Expression from PI is limited to adipose tissue and liver. Sequence comparison of the proximal promoters of ovine and mouse PIs demonstrates high nucleotide identity and that they are characterized by a TATA box at −29, C/EBP (CCAAT enhancer-binding protein)-binding motifs and multiple E-box motifs. A 4.3kb ovine PI-luciferase reporter construct is insulin-responsive when transfected into differentiated ovine adipocytes, whereas when this construct is transfected into ovine preadipocytes and HepG2 cells the construct is inactive and is not inducible by insulin. By contrast, transfection of a construct corresponding to 132bp of the proximal promoter linked to a luciferase reporter is active and inducible by insulin in all three cell systems. Insulin signalling to the −132bp construct in differentiated ovine adipocytes involves, in part, an E-box motif at −114. Upstream stimulatory factor (USF)-1 and USF-2, but not sterol regulatory element-binding protein 1 (SREBP-1), are major components of protein complexes that bind this E-box motif. Activation of the 4.3kb PI construct in differentiated ovine adipocytes is associated with endogenous expression of PI transcripts throughout differentiation; PI transcripts are not detectable by RNase-protection assay in ovine preadipocytes, HepG2 cells or 3T3-F442A adipocytes. These data indicate the presence of repressor motifs in PI that are required to be de-repressed during adipocyte differentiation to allow induction of the promoter by insulin.

scholarly journals The role of acetyl-CoA carboxylase phosphorylation in the control of mammary gland fatty acid synthesis during the starvation and re-feeding of lactating rats

1986 ◽  
Vol 237 (1) ◽  
pp. 85-91 ◽  
Author(s):  
M R Munday ◽  
D G Hardie
Keyword(s):  
Mammary Gland ◽  
Kinetic Parameters ◽  
Protein Phosphatase ◽  
Mammary Glands ◽  
Phosphate Content ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Carboxylase Activity

Activation of acetyl-CoA carboxylase during incubation of crude extracts of lactating rat mammary gland with Mg2+ and citrate can be blocked by NaF, suggesting that it represents a dephosphorylation of the enzyme. The greater extent of activation in extracts from 24 h-starved rats (200%) compared with fed controls (70%) implies that the decrease in acetyl-CoA carboxylase activity in response to 24 h starvation may involve increased phosphorylation of the enzyme. Acetyl-CoA carboxylase was purified from the mammary glands of lactating rats in the presence of protein phosphatase inhibitors by avidin-Sepharose chromatography. Starvation of the rats for 24 h increased the concentration of citrate giving half-maximal activation by 75%, and decreased the Vmax. of the purified enzyme by 73%. This was associated with an increase in the alkali-labile phosphate content from 3.3 +/- 0.2 to 4.5 +/- 0.4 mol/mol of enzyme subunit. Starvation of lactating rats for 6 h, or short-term insulin deficiency induced by streptozotocin injection, did not effect the kinetic parameters or the phosphate content of acetyl-CoA carboxylase purified from mammary glands. The effects of 24 h starvation on the kinetic parameters and phosphate content of the purified enzyme were completely reversed by re-feeding for only 2.5 h. This effect was blocked if the animals were injected with streptozotocin before re-feeding, suggesting that the increase in plasma insulin that occurs on re-feeding was responsible for the activation of the enzyme. The effects of re-feeding 24 h-starved rats on the kinetic parameters and phosphate content of acetyl-CoA carboxylase could be mimicked by treating enzyme purified from 24 h-starved rats with protein phosphatase-2A in vitro. Our results suggest that, in mammary glands of 24 h-starved lactating rats, insulin brings about a dephosphorylation of acetyl-CoA carboxylase in vivo, which may be at least partly responsible for the reactivation of mammary lipogenesis in response to re-feeding.

scholarly journals Polymer-protomer transition of acetyl-CoA carboxylase occurs in vivo and varies with nutritional conditions.

1980 ◽  
Vol 255 (21) ◽  
pp. 10033-10035
Author(s):  
B.A. Ashcraft ◽  
W.S. Fillers ◽  
S.L. Augustine ◽  
S.D. Clarke
Keyword(s):  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Nutritional Conditions

scholarly journals Growth-hormone-prolactin interactions in the regulation of mammary and adipose-tissue acetyl-CoA carboxylase activity and gene expression in lactating rats

1992 ◽  
Vol 285 (2) ◽  
pp. 469-475 ◽  
Author(s):  
M C Barber ◽  
M T Travers ◽  
E Finley ◽  
D J Flint ◽  
R G Vernon
Keyword(s):  
Adipose Tissue ◽  
Mammary Gland ◽  
Serum Prolactin ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Carboxylase Activity ◽  
Total Enzyme Activity ◽  
Mrna Concentration ◽  
Total Enzyme ◽  
Activation Status

The factors and mechanisms responsible for the reciprocal changes in lipogenesis in rat mammary gland and adipose tissue during the lactation cycle have been investigated. Lactation decreased the activation status and mRNA concentration of acetyl-CoA carboxylase in adipose tissue. Litter removal decreased the mRNA concentration of acetyl-CoA carboxylase in the mammary gland and increased the enzyme's mRNA concentration and activation status in adipose tissue. Lowering serum prolactin concentration in lactating rats decreased the amount of mammary acetyl-CoA carboxylase mRNA and increased that of adipose tissue, and increased the activation status of the enzyme in adipose tissue. Decreasing serum growth hormone (GH) alone had little effect on acetyl-CoA carboxylase in lactating rats, although it did lower pup growth rate and serum concentration of insulin-like growth factor-I. Lowering serum GH concentration exacerbated the effects of decreasing serum prolactin on mammary-gland (but not adipose-tissue) acetyl-CoA carboxylase mRNA and further increased the rise in activation status of the adipose-tissue enzyme induced by decreasing serum prolactin. Changes in acetyl-CoA carboxylase mRNA in both mammary and adipose tissue were paralleled by changes in total enzyme activity except after litter removal, when there was a disproportionately large decrease in total enzyme activity of the mammary gland. Thus prolactin has a major and GH a minor role in the regulation of acetyl-CoA carboxylase activity during lactation. Changes in mammary activity in response to prolactin and GH are primarily due to alterations in gene transcription, whereas adaptation in adipose tissue involves both changes in gene transcription and activation status.

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