Abstract
Stearoyl-CoA desaturase gene 1 (scd1) mRNA levels were induced during the 1st day of 3T3-L1 differentiation. scd1 expression had previously been observed only in late-stage differentiation, during the period of triacylglycerol accumulation. The induction of 3T3-L1 differentiation requires treatment with insulin, dexamethasone, and an agent that increases intracellular cAMP levels. Treatment of preadipocytes with cAMP-elevating agents caused an increase in scd1 mRNA concentrations. Insulin and dexamethasone had no effect on scd1 mRNA expression in preadipocytes. The increase in mRNA resulted from transcriptional activation of the scd1 gene. 8-Bromo-cAMP treatment of differentiated adipocytes had no effect on scd1 mRNA levels, suggesting a preadipocyte-specific effect. The increase in scd1 mRNA occurred maximally after 6 h of cAMP treatment and was shown to require protein synthesis. Deletion and mutagenesis analyses have localized the cAMP response to sequence within the first 250 base pairs of the 5'-flanking region of the scd1 gene. Treatment with phorbol ester enhanced the induction of scd1 mRNA by cAMP, suggesting the involvement of AP-2 as a mediator of the cAMP response. The induction of scd1 expression by cAMP during early differentiation was distinct from that observed during late adipocyte development. This early expression presents a novel regulated function of scd1 during the differentiation process, independent of its lipogenic role in late adipocyte development. The induction of scd1 also introduces a role for cAMP in 3T3-L1 differentiation.
Highlights
The 3T3-L1 preadipocyte cell line, originally obtained from mouse embryos [1], provides a reliable model for characterizing the events responsible for adipocyte differentiation [2]
One of the lipogenic genes known to be expressed during late differentiation is scd1.1 scd1 encodes for an isozyme of stearoylCoA desaturase, a key enzyme involved in the biosynthesis of unsaturated fatty acids and the regulation of fatty acid biosynthesis. scd1 catalyzes the ⌬9-cis desaturation of fatty acylCoAs, the major products being palmitoleoyl- and oleyl-CoA [4]
We have previously demonstrated that cAMP does not induce scd1 mRNA expression in mature adipocytes
Summary
Materials—Restriction enzymes and other nucleic acid modifying enzymes were obtained from Promega. Cell Culture—3T3-L1 preadipocytes were maintained and induced to differentiate as described [11]. Isolation of RNA and Quantitation of scd mRNA Levels—RNA was isolated from 3T3-L1 cells as described by Chirgwin et al [12]. Isolation of 3T3-L1 Preadipocyte Nuclei and in Vitro Run-on Transcription—The isolation of 3T3-L1 nuclei was performed as described by Love and Minton [15]. Transfection and Assay of Expression of 5Ј-Flanking Region Constructs—Proliferating (80% confluent) 3T3-L1 preadipocytes were either transiently or stably transfected using lipofectamine transfection reagent as described by the manufacturer (Life Technologies, Inc.). Total RNA was collected and analyzed by RNase protection using an scd1-specific complementary riboprobe. B, data for A were quantitated and plotted as fold induction of differentiated time points over undifferentiated cells.
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