Volume 25, Issue 1, Pages 86-92 (January 2017) Circadian Clock Interaction with HIF1α Mediates Oxygenic Metabolism and Anaerobic Glycolysis in Skeletal Muscle  Clara Bien Peek, Daniel C. Levine, Jonathan Cedernaes, Akihiko Taguchi, Yumiko Kobayashi, Stacy J. Tsai, Nicolle A. Bonar, Maureen R. McNulty, Kathryn Moynihan Ramsey, Joseph Bass  Cell Metabolism  Volume 25, Issue 1, Pages 86-92 (January 2017) DOI: 10.1016/j.cmet.2016.09.010 Copyright © 2017 Elsevier Inc. Terms and Conditions

Cell Metabolism 2017 25, 86-92DOI: (10.1016/j.cmet.2016.09.010) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Circadian Clock Controls Oxygen Consumption and Anaerobic Glycolysis through the Regulation of HIF1α (A) OCR from WT and Bmal1−/− C2C12 myotubes treated sequentially with oligomycin and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) (n = 9–10) is shown. (B) ECAR from intact WT and Bmal1−/− C2C12 myotubes treated sequentially with glucose and oligomycin (n = 10) is shown. (C) Immunoblots of HIF1α and β-actin in WT versus Bmal1−/− (left) and Cry1/2+/− versus Cry1/2−/− (right) MEFs exposed for 14 hr to increasing doses of CoCl2 (0 μM and a dilution curve from 7.8 to 125 μM) are shown. (D) Immunoblots of HIF1α and β-actin following exposure to 1% O2 for the indicated times in WT versus Bmal1−/− C2C12 myotubes are shown. (E) Expression of HIF target genes in WT versus Bmal1−/− C2C12 myotubes exposed to 1% O2 for 6 hr (n = 7–14) is shown. (F) Expression of HIF target genes in gastrocnemius muscle from adult life-inducible skeletal muscle Bmal1−/− mice (ACTA-rtTA-TRE-Cre;Bmalfx/fx) and controls (ACTA-rtTA-TRE-Cre and Bmalfx/fx) (n = 4–5) is shown. (G) Relative luciferase activity of C212 myoblasts transfected with HRE-LUC and plasmids expressing circadian and HIF TFs (n = 3). Data are represented as mean ± SEM (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001). See also Figures S1 and S2. Cell Metabolism 2017 25, 86-92DOI: (10.1016/j.cmet.2016.09.010) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 HIF Regulates Circadian Clock Function (A) Normalized photomultiplier detection of PER2:LUC reporter oscillations in synchronized C2C12 myotubes following exposure to 125 μM dimethyloxalylglycine (DMOG), an HIF1/2α stabilizer (green line), or untreated controls (black line) (n = 3) is shown. (B) ChIP of HIF1α and occupancy of the E-box-containing promoter regions of Per2 and Cry1, the HRE-containing promoter regions of Ldha and Vegfa, and the Slc2a2 gene promoter, which has no E-box or HRE (n = 3), are shown. (C) Relative luciferase activity of PER2:LUC in the presence of the indicated BMAL1 and HIF combinations in C212 myoblasts (n = 4) is shown. (D and E) Relative gene expression of clock target genes in (D) non-synchronized C2C12 myotubes exposed to 1% O2 for 6 hr versus normoxic conditions (n = 7–14) and (E) MEFs isolated from Cag-CRE-ER;Vhlfx/fx versus control mice (n = 4). Data are represented as mean ± SEM (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001). See also Figure S3. Cell Metabolism 2017 25, 86-92DOI: (10.1016/j.cmet.2016.09.010) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Clock and HIF Transcriptional Response to Strenuous Exercise Varies According to Time of Day in Skeletal Muscle WT mice were exercised by treadmill running to exhaustion at ZT0 (start of light period), ZT6 (mid-light period), ZT12 (start of dark period), or ZT18 (mid-dark period) prior to immediate extraction of gastrocnemius muscle and subsequent quantification of HIF and clock target mRNAs (n = 7). Data are represented as mean ± SEM (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 using two-way ANOVA statistical analysis). Cell Metabolism 2017 25, 86-92DOI: (10.1016/j.cmet.2016.09.010) Copyright © 2017 Elsevier Inc. Terms and Conditions