Temporal orchestration of circadian autophagy rhythm by C/EBPβ - PubMed (original) (raw)
Temporal orchestration of circadian autophagy rhythm by C/EBPβ
Di Ma et al. EMBO J. 2011.
Abstract
Temporal organization of tissue metabolism is important for maintaining nutrient and energy homeostasis in mammals. Autophagy is a conserved cellular pathway that is activated in response to nutrient limitation, resulting in the degradation of cytoplasmic components and the release of amino acids and other nutrients. Here, we show that autophagy exhibits robust circadian rhythm in mouse liver, which is accompanied by cyclic induction of genes involved in various steps of autophagy. Functional analyses of transcription factors and cofactors identified C/EBPβ as a potent activator of autophagy. C/EBPβ is rhythmically expressed in the liver and is regulated by both circadian and nutritional signals. In cultured primary hepatocytes, C/EBPβ stimulates the program of autophagy gene expression and is sufficient to activate autophagic protein degradation. Adenoviral-mediated RNAi knockdown of C/EBPβ in vivo abolishes diurnal autophagy rhythm in the liver. Further, circadian regulation of C/EBPβ and autophagy is disrupted in mice lacking a functional liver clock. We have thus identified C/EBPβ as a key factor that links autophagy to biological clock and maintains nutrient homeostasis throughout light/dark cycles.
Conflict of interest statement
The authors declare that they have no conflict of interest.
Figures
Figure 1
Rhythmic induction of autophagy in the liver. (A) Immunoblotting of liver lysates using indicated antibodies. Pooled samples from four mice were used for each time point. ZT0 and 12 represent the onset of light and dark cycles, respectively. The figure represents one of three independent sets of circadian samples. (B) Immunoblots showing LC3-II levels in the livers from mice injected with PBS (−) or leupeptin (+) 3 h before tissue harvest. Pooled samples from three mice were used for each lane. (C) Quantitation of in vivo autophagy flux. Following normalization to β-actin in (B), relative leupeptin-induced LC3-II accumulation was calculated by subtracting LC3-II levels in PBS-treated from leupeptin-treated mice for each time point. Data represent mean±s.d. of one representative experiment. (D) Transmission electron micrograph of liver sections at ZT5, 11, 17, and 23. The scale bar in the upper right corner of ZT5 figure represents two microns. The right lower corner shows the higher magnification highlighting a cytosolic region. Note the presence of double-membraned autophagosomes (arrow head). (E) Quantitation of autophagosome abundance in (D). Data represent mean±s.e. *P<0.000001 (ZT11 versus 17, _n_=10–16 cells). Student's _t_-test was applied.
Figure 2
Rhythmic induction of autophagy gene expression in the liver. (A) qPCR analysis of autophagy genes at different time points of mouse livers. Pooled samples from four mice were used for each time point. Data represent mean±s.d. of one of three independent studies. Becn1 serves as an example gene with modest diurnal oscillation at mRNA level. (B) Immunoblots of autophagy proteins in mouse livers at indicated time points. Pooled samples from four mice were used for each time point.
Figure 3
Induction of autophagy gene expression and autophagy process by C/EBPβ. (A) Clustering analysis of autophagy-related genes in primary hepatocytes transduced with GFP or C/EBPβ adenoviruses for 48 h. Blue and yellow represent low and high mRNA expression, respectively. (B) qPCR analysis of autophagy gene expression. Fold induction by C/EBPβ in primary hepatocytes 24 h (blue) or 48 h (red) following adenoviral transduction is shown. Data represent mean±s.d. of one representative experiment. (C) Protein expression of autophagy genes in hepatocytes transduced with GFP or C/EBPβ adenoviruses. Arrowheads point to two C/EBPβ isoforms generated from alternative translation start sites. (D) Immunoblots of total lysates from transduced primary hepatocytes treated with vehicle or concanamycin in triplicates. Blots with different exposure time were shown to illustrate LC3-I and LC3-II signals. (E) Quantitation of LC3-II protein levels following normalization to β-actin. Data represent mean±s.e. Student's _t_-test was applied. *P<0.01; **P<0.001. (F) Protein degradation assay in transduced hepatocytes in the absence (open) or presence of 3-MA (filled). Note that C/EBPβ-inducible proteolysis is sensitive to 3-MA treatment. Student's _t_-test was applied. *P<0.05.
Figure 4
C/EBPβ stimulates the transcription of autophagy genes through direct promoter occupancy. (A) Transcriptional assays using indicated promoter luciferase constructs in the presence of vector (open), 25 ng (grey), or 100 ng (filled) of C/EBPβ expression plasmid. Predicted C/EBPβ binding sites are indicated with solid bars. Data represent mean±s.e. Student's _t_-test was applied. *P<0.01 (vector versus 25 ng C/EBPβ); **P<0.001 (vector versus 100 ng C/EBPβ). (B) ChIP assay using control IgG (open) or C/EBPβ (filled) antibodies. Relative enrichment was determined by qPCR.
Figure 5
C/EBPβ expression is regulated by circadian and nutritional signals. (A, B) Hepatic C/EBPβ mRNA and protein expression at different time points. (C) Immunoblotting of liver lysates from fed, 24 h-fasted or 24 h-refed mice (after 24 h fast). Samples were collected at ZT4. (D, E) qPCR analysis of hepatic gene expression from fed, 24 h-fasted or 24 h-refed mice, the same group as in (C). Pooled samples from 3 to 5 mice were used for each data point. Data in (A), (D), and (E) represent mean±s.d.
Figure 6
Regulation of C/EBPβ and autophagy by restricted feeding. (A) The diagram indicates restricted feeding schedule and food availability (grey box, arrows indicate tissue harvest times). (B) qPCR analysis of hepatic gene expression in mice fed during dark (NF) or light (DF) phase. (C) Immunoblotting of liver lysates from mice undergoing restricted feeding. Pooled samples from 3 to 5 mice were used for each data point. Data in (B) represent mean±s.d.
Figure 7
Liver autonomous clock is required for normal autophagy rhythm. (A) qPCR analysis of hepatic genes in control (filled diamond) and liver-specific Bmal1 knockout mice (open square, Bmal1 LKO). (B) Immunoblots of total liver lysates in control and Bmal1 LKO mice using indicated antibodies. (C) In vivo autophagy flux assay was performed at ZT6-9. Pooled samples from 3 to 4 mice were used for each group. Data in (A) represent mean±s.d.
Figure 8
C/EBPβ is essential for physiological regulation of autophagy in the liver. (A) qPCR analysis of hepatic genes in mice transduced with control (Scrb) or siC/EBPβ (siC) adenoviruses under fed (open) and 16 h-fasted (filled) conditions. Samples were harvested at ZT19. (B) Immunoblots of liver lysates in mice from (A) under fed and fasted conditions. Pooled samples from 3 to 5 mice were used for each data point. Data in (A) represent mean±s.d.
Figure 9
C/EBPβ is essential for circadian autophagy regulation in the liver. (A) qPCR analysis of hepatic genes in mice transduced with control (Scrb, filled diamond) or siC/EBPβ (siC, open square) adenoviruses harvested at indicated time points. Data represent mean±s.d. (B) Immunoblots of liver lysates in mice from (A) at indicated time points. (C) In vivo autophagy flux assay was performed in mice transduced with control (Scrb) or siC/EBPβ (siC) adenoviruses at ZT6-9. Pooled samples from 3 to 5 mice per group were analysed. (D) Model depicting circadian autophagy regulation through C/EBPβ. Note that C/EBPβ receives both circadian and nutritional input and coordinately regulates the program of autophagy gene expression.
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