Circadian Clock Interaction with HIF1α Mediates Oxygenic Metabolism and Anaerobic Glycolysis in Skeletal Muscle

Abstract

Summary Circadian clocks are encoded by a transcription-translation feedback loop that aligns energetic processes with the solar cycle. Here we show that genetic disruption of the clock activator BMAL1 in skeletal myotubes and fibroblasts increased levels of the hypoxia-inducible factor 1α (HIF1α) under hypoxic conditions. Bmal1−/− myotubes displayed reduced anaerobic glycolysis, mitochondrial respiration with glycolytic fuel, and transcription of HIF1α targets Phd3, Vegfa, Mct4, Pk-m, and Ldha, whereas abrogation of the clock repressors CRY1/2 stabilized HIF1α in response to hypoxia. HIF1α bound directly to core clock gene promoters and, when co-expressed with BMAL1, led to transactivation of PER2-LUC and HRE-LUC reporters. Further, genetic stabilization of HIF1α in Vhl−/− cells altered circadian transcription. Finally, induction of clock- and HIF1α-target genes in response to strenuous exercise varied according to the time-of-day in wild-type mice. Collectively, our results reveal bi-directional interactions between circadian and HIF pathways that influence metabolic adaptation to hypoxia.

Metabolic Plasticity Enables Circadian Adaptation to Acute Hypoxia in Zebrafish Cells

“…As a consequence, the NAD + /NADH ratio oscillation aligns with the hypoxia induced high amplitude rhythm of the NADP + /NADPH ratio and nocturnal Prx ox , leading to highly oxidized states of the cells during the night periods. While oscillations of canonical clock genes are already dampened after short time hypoxic incubation as it is also known from experiments with chronic hypoxia [10, 14, 15, 28, 30], circadian rhythms of cytosolic H 2 O 2 remain absolutely unaffected by the hypoxia induced metabolic and transcriptional changes, implicating that a cellular timekeeping mechanism apart from the transcriptional clock is quite robust against the stress of reduced oxygen tensions. The hypoxia-induced and mostly Hif-1α driven response observed in cellular metabolic rhythms might thus form the basis for the observed hypoxia induced attenuation of the TTFL, against the background of the reduced binding affinity of the CLOCK/BMAL1 complex to DNA in a highly oxidized cellular environment.…”

“…The reciprocal interaction between the hypoxic signaling pathway and the circadian clock was described for the first time in the vertebrate model zebrafish [10, 28] and only recently also found and characterized in more detail in mammals [5, 14, 15], which stresses the translational potential of the findings and the essential nature of the observed link, representing a fundamental aspect of eukaryotic cell biology. In this context, reduced oxygen tensions were found to dampen oscillation amplitudes of core clock genes in vertebrates including mammals through direct binding of Hypoxia Inducible Factor 1α (Hif-1α) to E-boxes of the genes period1, period2 and cryptochrome1 , all members of the negative limb of the core transcriptional translational feedback loop (TTFL) [10, 14, 15, 28, 30].…”

“…Chronic hypoxia is known to reduce the oscillation amplitudes of specific clock genes [10, 14, 15, 28]. To confirm the hypoxia induced attenuation of the TTFL also for the present experimental setup using acute hypoxia we screened mRNA oscillation of core clock genes and found that expression levels of the clock genes period1b ( per1 ) and cryptochrome1aa ( cry1 ) were significantly reduced, as was the copy number of clock1a ( clock1 ) recorded in the early LD night (Fig.…”

“…In this context, reduced oxygen tensions were found to dampen oscillation amplitudes of core clock genes in vertebrates including mammals through direct binding of Hypoxia Inducible Factor 1α (Hif-1α) to E-boxes of the genes period1, period2 and cryptochrome1 , all members of the negative limb of the core transcriptional translational feedback loop (TTFL) [10, 14, 15, 28, 30]. In turn, the expression of Hif-1α protein was shown to be clock controlled, and examples for the physiological relevance of the observed link between both pathways was likewise illustrated in zebrafish [28] and in mice [5, 15].…”

“…Models for the obvious crosstalk between metabolic oscillations and the TTFL were proposed by several authors [24-26] and only recently the pentose phosphate pathway (PPP), the main source of NADPH production as reducing equivalent was identified as a major regulator of the TTFL [27]. The influence of hypoxia on the TTFL has been investigated in several vertebrate studies [5, 7, 10, 14, 15] and the HIF-1α mediated dampening of specific core clock gene oscillation amplitudes, first described in zebrafish [10, 28] was specified recently also in mice [14, 15]. Similarly, consequences of the reported link between both pathways for organismic physiology were illustrated in both models, in zebrafish [28] and in mice [5, 15], which stresses the translational impact of the findings and the essential nature of the mutual interaction between the circadian clock and oxygen metabolism.…”