Klf15 Orchestrates Circadian Nitrogen Homeostasis Darwin Jeyaraj, Frank A.J.L. Scheer, Jürgen A. Ripperger, Saptarsi M. Haldar, Yuan Lu, Domenick A. Prosdocimo, Sam J. Eapen, Betty L. Eapen, Yingjie Cui, Ganapathi H. Mahabeleshwar, Hyoung-gon Lee, Mark A. Smith, Gemma Casadesus, Eric M. Mintz, Haipeng Sun, Yibin Wang, Kathryn M. Ramsey, Joseph Bass, Steven A. Shea, Urs Albrecht, Mukesh K. Jain  Cell Metabolism  Volume 15, Issue 3, Pages 311-323 (March 2012) DOI: 10.1016/j.cmet.2012.01.020 Copyright © 2012 Elsevier Inc. Terms and Conditions

Cell Metabolism 2012 15, 311-323DOI: (10.1016/j.cmet.2012.01.020) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 Klf15 Exhibits 24-hr Periodicity and Is Driven by the Core Clock Machinery (A) Klf15 mRNA accumulation from WT mice livers (n = 5 per time point). (B) Representative KLF15 and U2AF65 protein expression from WT and Klf15 null liver nuclei. (C) KLF15 protein densitometry from three replicates. (D) Klf15-luciferase is induced in a dose-dependent fashion by CLOCK/BMAL1; inset illustrates four E-Box motifs in the Klf15 promoter (−5 kb). (E) Klf15 mRNA accumulation in WT and Bmal1 KO livers. (F) Rhythmic binding of BMAL1 on the Klf15 promoter (n = 3 per time point). Data presented as mean ± SEM. Cell Metabolism 2012 15, 311-323DOI: (10.1016/j.cmet.2012.01.020) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 Nitrogen Homeostasis Exhibits 24-Hour Periodicity, Driven by Klf15, in Mice (A–D) Plasma total AA pool, alanine, BCAA, and urea measured every four hours over a circadian period after placing mice in constant darkness for 38 hr (n = 5 per time point). The data are double-plotted, and ANOVA was used to determine rhythmicity. (E) Cumulative food intake measured every 5 min in WT and Klf15 null mice (n = 4 per group). (F) Total body weights of WT and Klf15 null mice (n = 4 per group). (G and H) Plasma total AA pool, urea from WT and Klf15 null mice measured every 4 hr under L/D and double-plotted to illustrate rhythmicity (n = 5 per group per time point). Data presented as mean ± SEM. Cell Metabolism 2012 15, 311-323DOI: (10.1016/j.cmet.2012.01.020) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 Klf15 Regulates Rhythmic Amino Acid Utilization Schematic illustrates the interorgan transport and utilization of AAs (i.e., the glucose-alanine cycle). (A and B) Skeletal muscle Alt and Bcat2 expression in WT and KLF15 null mice (n = 4 per group per time point). (C and D) Plasma alanine and BCAA in WT and Klf15 null mice (n = 5 per group per time point). (E) Plasma glucose in WT and Klf15 null mice (n = 5 per group per time point). (F) ChIP for KLF15 on Alt promoter (n = 3 per time point). (# p < 0.05 at all time points between WT and Klf15 null). Data presented as mean ± SEM. Cell Metabolism 2012 15, 311-323DOI: (10.1016/j.cmet.2012.01.020) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 Klf15 Regulates Rhythmic Nitrogeneous Waste Excretion Schematic illustrates the excretion of nitrogenous waste products (i.e., the urea cycle). (A and B) Plasma glutamate, ammonia in WT and Klf15 null mice (n = 5 per group per time point). (C) Otc expression in WT and Klf15 null livers (n = 4 per group per time point). (D) Plasma ornithine in WT and Klf15 null mice (n = 5 per group per time point). (E) OTC enzymatic activity measured from liver mitochondrial extracts from WT and KLF15 null mice (n = 4 per group). (F and G) Urinary levels of urea and ammonia in WT and KLF15 null mice (n = 5 per group). (H) ChIP for KLF15 on the Otc promoter (n = 3 per time point). (# p < 0.05 at all time points between WT and Klf15 null). (I–K)Results of neurobehavioral testing for (i) Y-maze, a test of working memory (n = 3 per group). (J and K) Fear conditioning during contextual changes (hippocampal function) and altered cues (amygdalar function) (n = 8 per group) (J), and Morris water maze test, a test of hippocampal function (n = 8 per group) (K). Data presented as mean ±SEM. Cell Metabolism 2012 15, 311-323DOI: (10.1016/j.cmet.2012.01.020) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 Feeding Alters Klf15 Expression and Nitrogen Homeostatsis (A–O) Following ad libitum feeding or feeding restricted to the light-phase (ZT3-ZT9) for one month: liver expression of Clock, Per2, and Cry1 (A–C); liver and skeletal muscle expression of Klf15 (D and E); total plasma AA, ammonia, and urea concentrations (F, G and H); skeletal muscle Alt and Bcat2 expression (I and J); liver expression of Otc (K); and plasma ornithine, alanine, BCAA, and insulin (L, M, N, and O) (n = 5 per group per time point). Data presented as mean ± SEM. Cell Metabolism 2012 15, 311-323DOI: (10.1016/j.cmet.2012.01.020) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 6 Wild-Type and Klf15 Null Adaptation to High-Protein Diet (A–E) Following 1 week of high-protein diet (HP; 70% casein) or normal diet (ND; 18% protein) WT and Klf15 null (n = 5 per group) blood glucose (A), plasma AAs (B), liver expression of Klf15, Alt, and Otc (C and D), plasma ammonia (D), and plasma urea (E). Data presented as mean ± SEM. Cell Metabolism 2012 15, 311-323DOI: (10.1016/j.cmet.2012.01.020) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 7 Human Endogeneous Circadian Nitrogen Homeostasis Fasting total plasma AA pool, alanine, BCAA, and urea exhibit endogenous circadian rhythmicity in humans during the forced desynchrony protocol. The cosine models (black lines) and 95% confidence intervals (gray areas) are based on mixed-model analyses and use precise circadian phase data. To show that these models adequately fit the actual data, we also plot the proportional changes across 60 circadian degree windows per individual, multiplied by the group average with SEM error bars (open circles with error bars). Data are double-plotted to aid visualization of rhythmicity. Lower x axis shows the circadian phase in degrees, with fitted core body temperature minimum assigned 0° and 360° equal to the individual circadian period (group average = 24.09 hr); top x axis shows corresponding average clock time for these individuals; vertical dotted lines show core body temperature minimum; horizontal gray bars show corresponding average habitual-sleep episode in the 2 weeks prior to admission. Cell Metabolism 2012 15, 311-323DOI: (10.1016/j.cmet.2012.01.020) Copyright © 2012 Elsevier Inc. Terms and Conditions