Ntu Library

Next, we investigated the effects of exercise on mRNA expression from either promoter.Wild type mice were allowed to run on incage running wheels for, or h, and mRNA expression was measured by qPCR.Strikingly, however, expression of PGC mRNA from the prox imal promoter was not induced at all, while expression from the alternative promoter increased fold.Western blot analysis of muscle extracts from sedentaryand exercised mice demonstrated strong induction of PGC. Hence, the increase in PGC expression seen after exercise stems entirely from the alternative promoter.Furthermore, the induction of PGC seen after an acute bout of exercise was at least in part dependent on adrenergic signaling.Six hours later, RNA was prepared from quadriceps muscles, and expression of PGC was measured by qPCR.Induction from the alternative promoter was induced fold, while expression from the prox imal promoter was not induced at all, paralleling the effects of exercise.Absolute expression from the alternative promoter is at baseline much less than expression from the prox imal promoter; after clenbuterol treatment, however, alternative expression surpasses that of the prox imal promoter. The induction of PGC from the alternative promoter was seen as early as h, peaked at h, and abated by hafter injection. Western blot analysis of muscle extracts from PBS and clenbuterolinjected mice demonstrated strong induction of PGC. Hence, as with exercise, adrenergic signaling induces expression of PGC specifically from the alternative promoter.PGC acts in an autoregulatoryloop to stimulate its own transcription. To test whether this autoregulatoryloop was activated by adrenergic stimulation, PGC MKO mice were injected with clenbuterol PGC transcripts were measured by qPCR with primers specific for either the alternative promoter UTR or the common UTR.PGC protein was absent in muscles from MKO mice, even after clenbuterol treatment. Hence adrenergic signaling on the PGC promoter is independent of PGC itself.This construct was then transfected by electroporation directly into the tibialis anterior muscle ofwild type mice.Hence, adrenergic stimulation activates the PGC alternative promoter invivo.In contrast, adrenergic activ ity had no ef fect on the prox imal PGC promoter. Comparison of the prox imal and alternative PGC promoters by sequence alignment did not reveal sign ificant similarities, suggesting that the two promoters did not arise by gene duplication.To test which sequences in the PGC alternative promoter render it susceptible to adrenergic stimulation, serial deletions were generated in the promoter construct.Electroporation of these plasmids into the tibialis anterior muscle, followed by measurement of luciferase activ ity, revealed that even a small deletion upstream of the transcriptional start site abrogated sensitiv ity to adrenergic signaling. The short region of the PGC alternative promoter identified above contains a consensus CRE, which is conserved across mammals. Mutation of this site almost abrogated the ability of clenbuterol to induce the PGC alternative transcript. Red and blue indicate elevated and reduced expression, respectively.This reprogramming is strikingly similar to that induced by transgenic expression of PGC in skeletal muscle, strongly suggesting a common mechanism.To test this notion, mice were treated with propranolol, a adrenergic receptor blocker, and then allowed to run on incage voluntarywheels.

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