This is the first study to investigate the relative influence of post-exercise cooling as well as the impact of reduced glycogen availability alone, and in combination, on promoter-specific mRNA expression of the transcriptional co-activator PGC-1α. Experiment 1 provided novel results demonstrating PGC-1α mRNA expression from the alternative promoter (Exon 1b) mirrors the pattern of expression for total PGC-1α mRNA previously described (Allan et al.
2017) (Fig.
2). Thus, the contribution to total PGC-1α mRNA expression post-exercise is likely a result of changes occurring from the alternative promoter (Exon 1b). Moreover, a systemic response to CWI was seen in the alternative promoter (Exon 1b) with large (~ 2000) fold changes from pre-exercise in the immersed and contralateral non-immersed limbs, versus a non-immersed control (< 1000-fold change from Pre) suggesting the response occurs independent of tissue temperature.
When muscle glycogen availability is very low (Experiment 2) (< 150 mmol·kg
−1dw; VLOW), the cold-induced augmented gene expression for total PGC-1α mRNA was abolished (Allan et al.
2019). New analysis of RNA samples herein focussing on the exon-specific response highlights VLOW glycogen limbs showing reduced mRNA expression via the alternative promoter (Exon 1b) versus the contralateral LOW glycogen limb, supporting the recent data indicating a glycogen threshold for adequate increases in gene expression to occur (Impey et al.
2018; Hearris et al.
2019). Moreover, a cold stimulus had no influence upon promoter-specific expression (Exon 1a and 1b) when glycogen concentration was low. Indeed, the combination of very low glycogen availability and post-exercise CWI showed the largest impaired response in gene expression of PGC-1α, irrespective of promoter region (Exon 1a and 1b). In Experiment 1, expression from the canonical promoter (Exon1a) showed a significant increase post-exercise in all limbs (two–fourfold change from Pre,
P < 0.05). Larger fold changes were present in expression from the alternative promoter (Exon1b), with values ranging ~ 1000–2000-fold increases from pre-exercise values (
P < 0.05). The sizeable difference in change of expression between the different promoter regions was likely due to the differential expression at pre-exercise (rest) and response to an exercise stimulus. Indeed, Exon 1a is constitutively expressed at higher levels basally (at rest) to a greater extent than Exon 1b, whilst Exon 1b is more responsive to an exercise stimulus (Martinez-Redondo et al.
2015; Popov et al.
2015b). In the present study, the pattern of response following exercise from the alternative (Exon 1b) promoter closely mirrored that of total PGC-1α gene expression seen in the same subjects previously (Allan et al.
2017), offering support to evidence showing the response of total PGC-1α mRNA to acute exercise is largely driven by the alternative promoter (Exon 1b) (Norrbom et al.
2011; Ydfors et al.
2013; Silvennoinen et al.
2015; Popov et al.
2015b). Additionally, new data herein to investigate the impact of commencing exercise with low glycogen availability combined with post-exercise cooling on the promoter-specific response (Experiment 2) show that PGC-1α alternative promoter (Exon 1b) expression continued to rise at 3 h post-exercise in all conditions, whilst expression from the canonical promoter (Exon 1a) decreased between the same time points (post-exercise–3 h post-exercise). This emphasises the role of the alternative promoter to the exercise-induced augmentation in total PGC-1α gene expression (Allan et al.
2017,
2019). Importantly, in Experiment 1, PGC-1α gene expression from the alternative promoter (Exon 1b) showed large increases in response to high-intensity intermittent cycling (CON, < 1000-fold change from Pre), which was augmented even further in limbs exposed to systemic cold stress (NOT and CWI, ~ 2000-fold change from Pre,
P = 0.07 and < 0.05 vs. CON, respectively). Indeed, the same pattern of response was observed in total PGC-1α mRNA (Allan et al.
2017), and the present data suggest that the contribution to cold-induced total PGC-1α gene expression is also driven by the alternative (Exon1b), and not the canonical (Exon1a) promoter region. These data are the first to show such a promoter-specific response to cold in animal or human studies.
In conclusion, this is the first study to show the systemic cold-induced augmentation of total PGC-1α as seen previously (Allan et al.
2017) is largely a result of increased expression from the alternative promoter (Exon 1b), rather than canonical promoter (Exon 1a). Evidence herein further supports the notion that PGC-1α gene expression from the alternative promoter (Exon 1b) is more reactive to an exercise stimulus than the canonical promoter region; with Exon 1b demonstrating lower levels at baseline, but more responsive to cold and exercise so demonstrating large fold increases. Whereas, Exon 1a was expressed more highly at rest and was less responsive to exercise and the cold and so demonstrates smaller fold increases. Moreover, commencing exercise with extremely low glycogen concentrations seems to negate the expected increase in PGC-1α gene expression irrespective of promoter region (Exon 1a and b), supporting the recent glycogen threshold hypothesis (Impey et al.
2018; Hearris et al.
2019). These data also provides greater support to the knowledge that post-exercise cooling may benefit important molecular signals of mitochondrial biogenesis, allowing confidence in the fact that endurance adaptation may be augmented following such modalities. Future research should look to include assessment of PGC-1α promoter-specific gene expression to allow greater mechanistic understanding of this important transcriptional co-activator.