Transcription of the acetyl-CoA carboxylase (ACC)-alpha gene is initiated from two promoters, promoter I (PI) and promoter II (PII) such that transcripts demonstrate heterogeneity in their 5' untranslated regions (UTR). Exons 1 and 2 (E1 and E2) are the primary exons in transcripts initiated from PI and PII respectively; E5 is the first coding exon present in all transcripts. In addition alternative exon splicing results in transcripts that either include or exclude a 47 nucleotide sequence corresponding to E4, such that E[1/4/5] and E[1/5] type transcripts result from PI activity, whereas transcripts containing E[2/4/5] or E[2/5] in the 5'UTR result from PII. In subcutaneous adipose tissue from non-pregnant non-lactating sheep approximately 60% of ACC-alpha transcripts are derived from PI, of which 85% are the E[1/5] type. Lactation resulted in an 88% reduction in total PI transcripts, of which the E[1/5] type was reduced 90% and the E[1/4/5] type 80%. By contrast lactation reduced the total levels of PII transcripts by only 50%. Culture of explants from the subcutaneous depot of lactating sheep with insulin plus dexamethasone for 72 h resulted in an 8-fold increase in both E[1/4/5] and E[1/5] types when compared with explants prior to culture. PII transcripts, by contrast, were increased 2-fold by culture in insulin plus dexamethasone and this was entirely attributed to an increase in the expression of the E[2/4/5] type. Dexamethasone acts to potentiate the action of insulin on PI and PII transcript abundance and this effect is greatest for PI transcripts. This study has demonstrated that repression of the ACC-alpha gene in adipose tissue during lactation is largely achieved through attenuation of PI transcript abundance and may be related, in part, to a change in the sensitivity of the apparatus that regulates PI transcript steady-state levels to insulin.
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MC Barber, L Pooley, and MT Travers
Expression of a variant acetyl-CoA carboxylase-alpha (ACC-alpha) mRNA encoding an isozyme either comprising (+24nt) or lacking (Delta24nt) an eight amino acid domain proximal to the Ser-1200 phosphorylation motif has been investigated in ovine and rat mammary tissue throughout pregnancy and lactation. The ratio of the Delta24nt mRNA: +24nt mRNA in ovine tissues varied from 0.1-0.25 (spleen, lung, muscle, heart, adipose tissue, brain) to 0.6-0.8 (pancreas, liver, kidney) to approximately 5.0 (lactating mammary gland). The sixfold increase in total ACC-alpha mRNA expression in mammary gland during lactation was due entirely to a tenfold increase in the level of the Delta24nt species as the level of expression of the +24nt species remained unaltered between pregnancy and lactation. This mode of expression of the +24nt and Delta24nt mRNAs was similar in rat mammary gland. Between day 20 of pregnancy and day 4 of lactation the ratio of the Delta24nt : +24nt mRNA increased from 2:1 to 10-20:1. Forced involution reduced the ratio of the two mRNAs to levels observed throughout pregnancy. Treatment of lactating rats with bromocryptine reduced the ratio of the Delta24nt : +24nt mRNA to relative levels observed after forced involution, suggesting that the exonic splicing responsible for the generation of the two mRNA isoforms is prolactin responsive.
MT Travers, RG Vernon, and MC Barber
We have investigated the mechanisms whereby lipogenesis is markedly suppressed in adipose tissue depots of lactating sheep. Expression of the gene encoding acetyl-CoA carboxylase (ACC), the flux-determining enzyme of the lipogenic pathway, is reduced approximately threefold in both omental and subcutaneous adipose tissue depots during late pregnancy and remains so into lactation when compared with non-pregnant, non-lactating animals. By comparison, total ACC enzyme activity in these adipose depots is suppressed approximately 25- to 30-fold in lactation. Culture of explants from the subcutaneous depot of lactating sheep with insulin plus dexamethasone for 72 h resulted in an approximately sevenfold increase in ACC mRNA, a fivefold increase in total enzyme activity and a marked increase in the proportion of the enzyme in the active state when compared with explants cultured with no added hormones for the same period. However, there was a lag of between 32 and 48 h before marked induction of any of these parameters by insulin plus dexamethasone was observed. Induction of the alpha-tubulin gene paralleled that of the ACC gene, suggesting that cytoskeletal rearrangements are associated with the aquisition of sensitivity to insulin plus dexamethasone. These results demonstrate that the reduction in lipogenic capacity in ovine adipose tissue during lactation is related to repression of the ACC gene, both at the level of steady-state mRNA abundance and possibly at translation, as well as to suppression of the mechanisms that regulate the proportion of ACC in the active state, and these are further related to the marked insensitivity of these parameters to insulin plus dexamethasone in vitro.