Regulation of protein and mRNA expression of the mTORC1 repressor REDD1 in response to leucine and serum

Expression of the mTORC1 repressor, Regulated in DNA Damage and Development 1 (REDD1), is elevated in skeletal muscle during various catabolic conditions including fasting, hindlimb immobilization, and sepsis. Conversely, REDD1 expression is suppressed by anabolic stimuli such as resistance exercise or nutrient consumption following a fast. Though it is known that nutrient consumption reduces REDD1 expression, it is largely unknown how nutrients and hormones individually contribute to the reduction in REDD1 expression. Therefore, the purpose of the present study was to determine how nutrients and hormones individually regulate REDD1 expression. HeLa cells were deprived of leucine or serum for 10 hours, after which either leucine or serum was reintroduced to cell culture medium for 60 minutes. Re-supplementation of either leucine or serum resulted in a reduction in REDD1 protein levels by 34.8 ± 5.8% and 54.1 ± 3.4%, respectively, compared to the deprived conditions. Re-supplementation of leucine or serum to deprived cells also led to a reduction in REDD1 mRNA content by 49.1% ± 2.7% and 65.0 ± 1.4%, respectively, compared to the deprived conditions. Interestingly, rates of REDD1 protein degradation were unaffected by either leucine or serum re-supplementation, as assessed in cells treated with cycloheximide to block protein synthesis. Likewise, addition of leucine or serum to cells treated with Actinomycin D to inhibit gene transcription failed to alter the rate of REDD1 mRNA degradation. The data indicate that the leucine or serum-induced suppression of REDD1 expression occurs independent of changes in the rate of degradation of either the REDD1 protein or mRNA. Thus, the leucine- or serum-induced suppression likely occurs through alternative mechanism(s) such as reduced REDD1 gene transcription and/or mRNA translation.