Insulin changes Bmal1 protein levels after gene activation to influence the liver’s daily clock

Previous reports from several groups have observed 8–12 h phase advances of Bmal1 mRNA rhythm before its target gene expression (Dbp and Nr1d1) in mouse liver22,23,24. Such unusually long delay between the expression of a transcription factor and its regulated genes suggests that Bmal1 transcriptional activity may be post-transcriptionally modulated. This notion is further supported by the fact that the fluctuations of Bmal1 nuclear accumulation and DNA-binding activity are uncoupled from its total protein oscillation in the liver22,24,25, whereas the underlying mechanism remains elusive. We observed consistent results that total Bmal1 protein amounts peaked around 8 h ahead of its nuclear levels (delayed from ZT20 to ZT4) and 12 h before its binding with Dbp promoter and subsequent induction of Dbp expression (delayed from ZT20 to ZT8) in livers of ad libitum fed mice (Supplementary Fig. 1a–c). Interestingly, we noticed that Bmal1 protein contains three consensus recognition motifs (at Ser 42, 422 and 513) for Ser/Thr kinase Akt, a key mediator of insulin signalling (Supplementary Fig. 1d). Considering that insulin has been reported to affect clock gene expression26,27 and its circulating levels also peaked around ZT20 (Supplementary Fig. 1e) when the oscillation of nuclear Bmal1 proteins reached the trough, we wondered whether insulin may block Bmal1 nuclear accumulation even with the peak of its total protein levels (Supplementary Fig. 1f). Indeed, Bmal1 protein amounts were significantly reduced in the nuclei but increased in the cytosol by insulin treatment in human hepatoma HepG2 cells or primary hepatocytes, as demonstrated by immunoblotting or immunostaining results (Fig. 1a,b), whereas insulin had little effect on the nucleus-cytosol shuttling of other key clock members (Clock, Cry1 and Per1, Fig. 1a). Moreover, the immunostaining results also showed that leptomycin B (LMB), a specific nuclear protein export inhibitor, abolished the inhibitory effect of insulin on nuclear Bmal1 accumulation (Fig. 1b), which was further verified by cell fraction analysis (Fig. 1c). Altogether, these data demonstrate that insulin modulates the subcellular localization of Bmal1 proteins in vitro.

We then examined whether insulin exerts the similar effect on hepatic Bmal1 in vivo. As mice usually do not eat much and their plasma insulin levels arrive at the nadir during light time, we fasted mice from ZT0 to prevent them from eating to disturb the results, and then intraperitoneally injected these animals with insulin at ZT6 and killed them after 0.5 to 6 h as indicated in Supplementary Fig. 1g. As expected, insulin markedly reduced nuclear Bmal1 protein accumulation, correspondingly increased its cytosolic distribution, and mildly enhanced its total amounts in mouse liver (Fig. 1d). As a result, insulin injection significantly attenuated Bmal1 occupancy on the promoters of its target genes (Dbp and Nr1d1, Fig. 1e) and subsequently their transcription (Fig. 1f). In line with previously reported in vitro results19, we also found that insulin injection increased hepatic mRNA levels of Per1 and Per2, the other two Bmal1-target genes (Supplementary Fig. 1h). As Bmal1 deficiency has been shown to enhance the expression of these two genes in mouse liver23,24, our data strongly indicate that insulin suppresses Bmal1 transcriptional activity. Meanwhile, Bmal1 expression was slightly reduced by insulin injection in the early stage of this experiment, whereas significantly increased at ZT12 (Supplementary Fig. 1g), which may be caused by the decrease of Nr1d1 that feedback inhibits Bmal1 transcription8. By contrast, no obvious difference in nuclear Bmal1 amounts and Dbp expression was observed in livers collected from the similar experiments performed during dark time (Supplementary Fig. 1i,j), when circulating insulin levels are high. Moreover, the inhibitory effect of insulin on Dbp expression was abolished in Bmal1 null primary hepatocytes (Fig. 1g), suggesting such insulin effect is mediated by Bmal1. In summary, our results indicate that insulin affects Bmal1 intracellular localization to limit its transcriptional activity in mouse liver.

As mentioned above that Bmal1 contains three consensus recognition motifs for Akt (Supplementary Fig. 1d), we wondered whether Akt is capable of phosphorylating Bmal1, which may be involved in insulin regulation of Bmal1. As expected, exposure of primary hepatocytes to insulin increased the phosphorylation of Bmal1, as detected by immunoblot assay with a phospho-Akt substrate antiserum (Fig. 2a), which was diminished in primary hepatocytes with the deficiency of Akt2, the major Akt isoform expressed in the liver (Fig. 2b). In addition, the results of co-immunoprecipitation assay showed the direct interaction between Bmal1 and Akt2 (Fig. 2c). We then performed liquid chromatography tandem mass spectrometry to characterize residue(s) in Bmal1 that undergo insulin-induced phosphorylation. This analysis revealed one single site Ser42 on Bmal1 (Supplementary Fig. 2a), which is highly conserved in vertebrates (Supplementary Fig. 2b). Consistent with mass spectrometry results, constitutive active Akt2 (Akt2-CA) induced wild-type (WT) Bmal1 protein phosphorylation, which was abolished by a serine to alanine mutation at Ser42 residue (S42A) but not a S422A/S513A double mutation, as shown by immunoblot assay with phospho-Akt substrate antiserum (Fig. 2d). Similarly, exposure of hepatocytes to insulin increased the phosphorylation of WT Bmal1, but not the S42A mutant (Supplementary Fig. 2c,d).

To specifically detect the phosphorylation of Ser42 on Bmal1, we generated a Ser42-phosphorylation-site-specific antibody (pSer42-Bmal1) with the epitope of R₃₇KRKGSATDYQE₄₈. We first confirmed its specificity by competitive inhibition studies with phospho or non-phosphopeptides in immunoblotting assays (Supplementary Fig. 2e). By using this antibody, we found that insulin significantly stimulated Ser42-phosphorylated Bmal1 accumulation in the liver (Fig. 2e and Supplementary Fig. 2f) as well as WT primary hepatocytes, which was markedly reduced in primary hepatocytes isolated from Akt2 knock-out mice or treated with PI3K inhibitor, LY-294002 (Fig. 2f, Supplementary Fig. 2g). The effect of Akt on Bmal1 appears direct as recombinant Akt protein derived from Sf9 cells was able to phosphorylate WT but not S42A mutant Bmal1 in vitro (Fig. 2g).

As a previous work has reported that S6K kinase is also capable of phosphorylating Bmal1 on Ser42 (ref. 28), we then checked whether Akt is the key mediator for insulin induction of Bmal1-Ser42 phosphorylation. In contrast to expectedly suppressing insulin-promoted Ser42 phosphorylation of Bmal1 proteins, the inhibition of S6k kinase activity by rapamycin treatment actually enhanced the amounts of pSer42-Bmal1 (Fig. 2h), which may be due to the loss of the negative-feedback regulation of S6k on insulin receptor substrate proteins29. In summary, our data indicate that insulin stimulates Akt-mediated phosphorylation of Ser42 on Bmal1.

As the above data suggest that insulin may limit nuclear Bmal1 accumulation via Akt-mediated Ser42 phosphorylation of Bmal1, we first verified whether Akt is involved in the regulation of Bmal1 subcellular distribution in vivo by checking the circadian pattern of nuclear Bmal1 protein in livers of Akt2 null mice. As expected, Akt2 deficiency significantly enhanced nuclear Bmal1 accumulations around ZT16–20, which results in the disruption of its nuclear rhythm (Fig. 3a and Supplementary Fig. 3a) and the increase of its target gene expression (Dbp and Nr1d1) at multiple time points (Supplementary Fig. 3b), even though these animals and WT littermates exhibited comparable rhythms of food intake (Supplementary Fig. 3c). Complementarily, no significant differences in nuclear Bmal1-S42A protein amounts were observed between the livers of Akt2 null and WT mice killed around ZT20 (Supplementary Fig. 3d). In line with the in vivo results, overexpression of Akt2-CA strikingly lowered Bmal1 protein abundance in the nucleus in primary hepatocytes (Fig. 3b and Supplementary Fig. 3e).

It is well established that AKT recognition sequences often overlap with putative binding sites of 14-3-3 proteins that affect subcellular localization of AKT-substrate proteins via a direct association30,31. Thus, we wondered whether phosphorylated Bmal1 interacts with 14-3-3. Indeed, insulin significantly enhanced the amounts of 14-3-3β proteins recovered from primary hepatocyte lysates by an anti-Bmal1 antibody, which was diminished by the pretreatment of these lysates with lambda phosphatase (Fig. 3c). Consistently, intraperitoneal injection of insulin enhanced the interaction between Bmal1 and 14-3-3β in the liver (Fig. 3d). Moreover, Bmal1-S42A mutation diminished insulin-induced Bmal1-14-3-3β interaction in primary hepatocytes (Fig. 3e), suggesting the necessity of Ser42 phosphorylation in this regards. Interestingly, the Akt/14-3-3-recognition motif of Ser42 (R₃₇KRKGS₄₂) overlaps with one of previously characterized nuclear localization signal motif (N₃₆RKRKG₄₁) on Bmal1 (ref. 32), which implies that Ser42 phosphorylation and subsequent interaction with 14-3-3β may affect Bmal1 localization. Supporting this notion, Bmal1-S42A mutant proteins resisted to the nuclear exclusion promoted by insulin treatment (Fig. 3f) or Akt2-CA overexpression (Fig. 3g and Supplementary Fig. 3f), compared with its WT proteins in primary hepatocytes. Taken together, these data suggest that insulin-Akt-14-3-3 signalling pathway plays a pivotal role in modulating Bmal1 subcellular distribution, which requires the phosphorylation of its Ser42 residue.

During studying the effect of insulin signalling on Bmal1 subcellular localization, we also noticed that total Bmal1 protein amounts were enhanced by insulin injection (Fig. 1d), but reduced by Akt2 deficiency (Fig. 3a and Supplementary Fig. 2h), suggesting the involvement of insulin-Akt signalling in the regulation of Bmal1 protein stability. This notion was further supported by the in vitro results that S42A mutation unstabilized Bmal1 (Supplementary Fig. 3g), whereas Akt2-CA increased Bmal1-WT protein amounts in HEK 293T cells (Supplementary Fig. 3h). Since Bmal1 deficiency has been reported to disrupt insulin-induced phosphorylation of Akt33, we further confirmed the necessity of Bmal1 for insulin inhibitory effect on Dbp expression (Fig. 1g) by using an adenovirus of Akt2-CA. In the absence or presence of insulin treatment, overexpression of Akt2-CA expectedly reduced Dbp expression to the comparable levels in insulin-treated WT hepatocytes, but exerted little effect in Bmal1 null hepatocytes (Supplementary Fig. 3i).

Next, we explored the effect of Akt-mediated Ser42 phosphorylation on Bmal1 transcriptional activity. With their PAS domains, Bmal1 and Clock form heterodimers that bind to E-box enhancer elements in the promoters of target genes by their basic helix-loop-helix domains34. Since Ser42 is close to the basic helix-loop-helix domain and far away from the two PAS domains on Bmal1, we hypothesized that phosphorylation of Ser42 may affect Bmal1 affinity to DNA but not Clock. Indeed, the results of chromatin immunoprecipitation assays showed that the amounts of Bmal1-S42A proteins recruited to E-box in the promoters of Dbp and Nr1d1 were three to fourfold higher than those of WT Bmal1 (Fig. 4a, left), even with less nuclear protein amounts (Fig. 4a, right), which suggests that phosphorylation of Ser42 disrupts Bmal1 binding to DNA. By contrast, S42A mutation did not affect Bmal1 associated with Clock in the absence of insulin, as determined by co-immunoprecipitation (Co-IP) assay (Supplementary Fig. 3j). As a result, Bmal1 mutants containing S42A induced much higher Per1-Luc activity than that of WT Bmal1 with co-expression of Clock in HEK 293T cells and neither S422A nor S513A mutation exhibited similar effects (Fig. 4b), suggesting a potential role of Ser42 phosphorylation in suppressing basal transcriptional activity of Bmal1. Supporting this notion, adenoviral expression of Bmal1-S42A mutant significantly increased the mRNA levels of Bmal1-target genes (Dbp and Nr1d1) in primary hepatocytes treated with insulin, compared with Ad-Bmal1-WT (Fig. 4c). In line with these in vitro results, the insulin-induced reduction of hepatic protein amounts of Dbp and Nr1d1 was reversed in mice tail-vein injected with Ad-Bmal1-S42A viruses (Fig. 4d). Taken together, these results suggest the importance of Ser42 phosphorylation in modulation of Bmal1 transcriptional activity.

Food availability is a potent synchronizer for the peripheral clock, as modification of feeding cycles by food entrainment resets the hepatic clock but exerts little impact on master clock in the SCN2,3,4. Tightly associated with feeding status, the temporal signalling of insulin is considerably altered during food entrainment. It thus comes as no surprise to speculate that insulin signal is involved in the regulation of the peripheral clock from the beginning of the change of feeding times. This notion is supported by several reports18,19,20,21, however, much less is known about the underlying mechanism. As the cycle of feeding periods is reversed from the start of daytime restricted feeding and we have shown the role of insulin signal in regulating Bmal1 transcriptional activity, we wanted to examine whether the dynamics of nuclear Bmal1 is altered correspondingly in mouse liver. Mice were killed every 4 h over the first 72 h under restricted feeding (RF) initiated at ZT12 or ad libitum feeding (AF) as control (Supplementary Fig. 4a). RF expectedly reversed the phase of nuclear Bmal1 protein oscillation from the beginning of Day1 and in the following 2 days (Fig. 5a and Supplementary Fig. 4b), which was disrupted in Akt2 knock-out mice (Fig. 5b and Supplementary Fig. 4c). Consequently, RF initiated the phase shifting of Bmal1-target gene expression (Dbp and Nr1d1) from Day1-ZT12 (Fig. 5c), whereas the phase of Bmal1 mRNA rhythm had not been altered until Day2 (Fig. 5c), which is mainly due to the change of Nr1d1 mRNA oscillation that inhibits Bmal1 expression8.

Interestingly, the peak of diurnal insulin oscillation was elevated gradually from an AF comparable level during Day1 RF (1.66±0.13 ng ml⁻¹ at RF-Day1-ZT8 versus 1.66±0.53 ng ml⁻¹ at AF-Day1-ZT20) to a strikingly high level in Day3 RF (10.01±0.91 ng ml⁻¹ at Day3-ZT4, Fig. 5d). To understand how RF causes such high insulin levels, we measured food consumption in these animals. RF did not significantly change the daily food intakes during the first two days until Day3 (Supplementary Fig. 4d), suggesting that RF mice were trained to learn the food shortage during dark time and prepare for it by consuming much more amounts of food during the feeding period than AF mice did. Supporting this notion, RF significantly increased the mouse stomach sizes and weights at ZT4, 8 and 12 in each day (Fig. 5e and Supplementary Fig. 4e) and reduced the amplitude of plasma leptin oscillation (Fig. 5f), compared with AF. By contrast, no significant change of the plasma levels of glucagon, which has been reported to affect Bmal1 transcription15, was observed at Day3-ZT16 and ZT4 (Supplementary Fig. 4f) when insulin levels were strikingly different between AF and RF mice, which may be due to a relatively short fasting period and food overconsumption during feeding times. Altogether, these data demonstrate a role of insulin in the regulation of the hepatic clock via modulating Bmal1 subcellular localization in food-entrained mice.

Next, we examined the importance of Ser42 phosphorylation in Bmal1 regulating the hepatic clock under either AF or RF conditions. As expected, the mRNA levels of Dbp and Nr1d1 were significantly enhanced in livers collected around either ZT4 or ZT20 from AF mice tail-vein injected with Ad-Bmal1-S42A, compared with those with Ad-Bmal1-WT (Fig. 6a). Consistently, hepatic Bmal1-S42A proteins resisted to feeding-induced nuclear exclusion around ZT4 and ZT8 in Day1-RF mice, compared with Bmal1-WT proteins (Fig. 6b and Supplementary Fig. 4g), which resulted in the enhancement of Dbp and Nr1d1 expression during feeding times (ZT4–ZT8, Fig. 6c). In line with the Akt2 knock-out data (Supplementary Fig. 3b), Ad-Bmal1-S42A had little effect on the phases of these two gene oscillations (Fig. 6c), which suggesting that multiple mechanisms contribute to the diurnal oscillations of clock-controlled genes in the liver.

The Akt2^−/− mouse line was generously provided by Dr Zhongzhou Yang (Laboratory of Heart and Disease Model, Model Animal Research Institute, Nanjing University, Nanjing, China); the Alb-Cre mouse line was generously provided by Dr Yong Liu (Institute for Nutritional Sciences, China); C57BL/6 WT mice were purchased from Shanghai Laboratory Animal Center, China; and the Bmal1^fl/fl mouse line was purchased from Jackson Laboratories. The Bmal1^fl/fl mouse line was crossed with Alb-Cre line to generate Bmal1 liver-specific knock-out mice. The 8- to 12-week old male mice were used in all the experiments and housed in the animal facility at Shanghai Institutes for Biological Sciences (SIBS). Mice were maintained on a 12-h light/12-h dark cycle for at least 2 weeks before study, had free access to water and regular diet and were randomly allocated into treatment groups. The number and strain of mice used in each experiment were specified in the corresponding figure legend. For RF experiments, food was removed 15 min before darkness and provided 15 min after light. After killing mice, blood and liver samples were collected. Plasma insulin levels were measured by ELISA kits (Millipore). The investigators were not blinded to allocation during experiments and outcome assessment. All animal care and use procedures were ethically approved by the Animal Care and Use Committee of Shanghai Institutes for Biological Sciences.

Plasmid expressing mouse Bmal1, Bmal1 (S42A), Bmal1 (S422A), Bmal1 (S513A), Bmal1 (S422A/S513A), Clock and Akt2 were constructed from mouse cDNA with specific primers as shown in Supplementary Table 1. Per1-Luc has been described previously43. pcDNA-Myr-FLAG-Akt2 (Akt2-CA) was constructed according to previously described44. Ad-FLAG-tagged Akt2-CA, Ad-FLAG-tagged WT or S42A mutant Bmal1 adenoviruses were constructed by using AdEasy system (Agilent Technologies). GFP adenovirus has been described previously45. In all, 1 × 10⁸ plaque-forming units (p.f.u.) of Bmal1-WT or 1.2 × 10⁸ p.f.u. of Bmal1-S42A adenoviruses were delivered to male C57BL/6J mice by tail-vein injection. Mice were killed 7 days after injection.

HEK 293T and HepG2 cells were purchased from ATCC, cultured in DMEM supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin and 1% l-glutamine and checked for mycoplasma by PCR or ELISA. For luciferase reporter assay, HEK 293T cells were transfected using 50 ng of Per1-Luc and 20 ng of β-gal, together with 100 ng of Bmal1 and Clock plasmids per well. Co-transfections were performed with a constant amount of DNA by adding 100 ng of the empty vector pcDNA3 (Invitrogen) and luciferase activity was measured. Mouse primary hepatocytes were prepared, cultured and infected with adenoviruses. Briefly, the hepatocytes were isolated by using collagenase (Sigma) and plated on dishes coated with rat tail tendon collagen (Sigma) according to manufacturers' instructions. Before experiments, all the cells were synchronized by 12 h serum starvation. For experiments with LY-294002 (Sigma, LY) or LMB (Cell Signaling Technology, LMB), hepatocytes were pre-treated with LY (10 μM) or LMB (20 ng ml⁻¹) for 2 or 5 h, before incubation with insulin (50 nM) for indicated times.

HEK 293T cells were cultured in 24-well plates, collected after 24 h of transfection and then lysed by adding 150 μl per well of the Gly-gly lysis buffer (25 mM Gly-gly, pH 7.8; 15 mM MgSO₄; 4 mM EGTA, pH 7.8; 1 mM DTT and 1% V/V Triton X-100). The plates were shook gently for 20 min by transference decoloring shaker, 50 μl per well of lysate was transferred to a well of a black 96-well plate, and then mixed with 50 μl of the luciferase assay mixture (20 mM Gly-gly, pH 7.8; 12 mM MgSO₄; 3 mM EGTA, pH 7.8; 0.2 mM potassium phosphate, pH 7.8; 2 mM ATP; 1.5 mM DTT and 1.25 mg ml⁻¹ firefly luciferin). Fluorescence intensity was measured by using Luninoscan Ascent (Thermo). For β-gal assay to normalize the luciferase assay results, another 50 μl per well of lysate was taken to be mixed with 50 μl per well of β-gal solution, incubated at room temperature until colour developed and then read at A420 on a plate reader (Epoch Microplate Spectrophotometer, BioTek).

Total RNAs from whole livers or primary cultured hepatocytes were extracted with Trizol (Invitrogen) and reverse-transcribed into cDNAs by prime script RT regent kit with gDNA eraser (Takara) according to the manufactures' instructions. Relative mRNA levels were determined by SYBR-green PCR kit with ABIPRISM 7900HT Sequence detector (Perkin Elmer) with specific primers as shown in. Ribosomal L32 mRNA levels were used as internal control. Supplementary Table 2

For immunoblot analysis, cultured cells or collected liver tissues were lysed in RIPA buffer containing proteinase inhibitors (PMSF, Cocktail and Phosphatase Inhibitors), followed by centrifuging at 12,000 r.p.m. for 15 min at 4 °C. Supernatants were collected and protein concentrations were determined by using BCA protein assay kit (Pierce, 23225) according to the manufactures' instructions. Identical amounts of proteins were subjected to SDS-PAGE electrophoresis and transferred to methanol-activated PVDF membranes (Millipore), which were blocked with 5% milk for 1 h at room temperature, incubated overnight with corresponding primary antibodies at 4 °C and then with secondary antibodies for 1 h at room temperature. Finally, the protein bands were developed by using ECL western blotting substrate (Thermo Pierce) and visualized by western film processor. Primary antibodies used in the experiments were as followed: Bmal1 (Abcam, catalogue No. ab3350, diluted 1:1,000); Clock (Abcam, catalogue No. ab3157, diluted 1:1,000); Histone H3 (Cell Signaling Technology, catalogue No. #9715, diluted 1:4,000); pSer473-Akt (Cell Signaling Technology, catalogue No. #9271, diluted 1:1,000); Akt (Cell Signaling Technology, catalogue No. #9272, diluted 1:1,000); FLAG-HRP (Sigma, M2, catalogue No. A8592, diluted 1:4,000 for immunoblotting Flag-tagged proteins); 14-3-3β (Santa Cruz Biotech, K-19, catalogue No. sc-629, diluted 1:3,000); Phospho-Akt Substrate (Cell Signaling Technology, 110B7E, catalogue No. #9614, diluted 1:1,000); β-Actin (Cell Signaling Technology, catalogue No. #4967, diluted 1:5,000) and α-Tubulin (Cell Signaling Technology, catalogue No. #2144, diluted 1:5,000). Uncropped blots are shown in. Supplementary Fig. 5

For immunoprecipitation analysis, liver tissues or cultured primary hepatocytes were lysed in the immunoprecipitation buffer (IP, 25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% NP-40, 1% Triton X-100, 10% glycerol and proteinase inhibitors), followed by centrifuging at 12,000 r.p.m. for 15 min at 4 °C. Supernatants were collected and their protein concentrations were adjusted to 1 μg μl⁻¹ with the BCA protein assay results. In all, 400–600 μl of each remaining homogenate was incubated with indicated antibodies together with IP-buffer-washed protein A agarose beads (16–156, Merck Millipore) overnight or with anti-FLAG M2 Magnetic beads (Sigma) for 2 h at 4 °C. The immunoprecipitation beads were then washed with IP buffer for six times, and subjected to immunoblotting analysis.

For in vitro kinase assay, FLAG-tagged WT or S42A Bmal1 proteins were immunoprecipitated from HEK 293T cells transfected with the corresponding plasmids. The proteins were incubated with 20 ng μl⁻¹ purified GST-Akt (Biovision) in the kinase buffer (25 mM Tris-HCl, pH 7.5, 5 mM β-Glyverophosphate, 2 mM DTT, 0.1 mM Na₃VO₄, 10 mM MgCl₂, 200 μM ATP) at 30 °C for 30 min. The reaction was stopped by boiling samples in SDS-PAGE loading buffer and then separated by SDS-PAGE. Phosphorylation signals were detected by using anti-phospho-Ser42-Bmal1 antibody (1:500).

Liver tissues or primary hepatocytes were cross-linked with 1% formaldehyde. Bmal1 or FLAG antibodies were used for immunoprecipitations along with rabbit IgG for negative controls. After removing crosslinks, DNA was extracted by using phenol–chloroform and precipitated with ethanol. Target promoters were analysed by using quantified SYBR-green real-time PCR and normalized to input chromatin signals with primers as shown in. Supplementary Table 3

Hepatocytes or liver tissues were washed two times with ice-cold PBS and pelleted at 3,000 r.p.m. at 4 °C. Pellets were resuspended in buffer containing 0.3% NP-40 for cells and 0.3% Triton X-100 for liver tissues in PBS with protease inhibitors. Cells/tissues were lysed with a dounce homogenizer and centrifuged at 4,200 r.p.m. for 10 min at 4 °C. Cytosolic supernatants were collected. Nuclear pellets were washed and resuspended in 200 μl nuclear extraction buffer (20 mM Tris, pH 7.4, 450 mM NaCl, 1% NP-40, 1 mM DTT, with protease inhibitors). Samples were sonicated and centrifuged at 13,000 r.p.m. for 20 min before collection of nuclear supernatants.

Primary cultured hepatocytes were pre-synchronized by 12 h serum starvation. After treatment, hepatocyte cells were fixed in 4% paraformaldehyde for 20 min, permeabilized with 0.3% triton X-100 and incubated with an anti-Bmal1 (1:1,000 dilution) or anti-FLAG antibody (1:1,000 dilution) for endogenous Bmal1 or FLAG-Bmal1 detection, respectively. Slides were washed and mounted with coverslips by using Vectashield mounting media containing 4, 6-diamidino-2-phenylindole.

Mass spectrometry studies were performed to identify the Bmal1 phosphorylation residues. Briefly, primary hepatocytes were infected by the FLAG-tagged Bmal1 adenovirus and then treated with or without insulin (50 nM) for 30 min. Immunoprecipitates of FLAG-Bmal1 proteins were separated by 10% gradient SDS-PAGE gels and stained with Coomassie Brilliant Blue for 15–20 min followed by de-staining at room temperature for 2 h. Entire lanes were cut into pieces and sent to Shanghai Applied Protein Technology Co. Ltd (China) for liquid chromatography-mass spectrometry (LC-MS/MS) analysis with the equipment of LTQ VELOS (Thermo Finnigan, San Jose, CA). The principle of peptide spectrum match was applied to characterize the phosphorylation residue on Bmal1.

Results were represented as mean±s.e.m. Statistical tests were selected based on appropriate assumptions with respect to data distribution and variance characteristics. The comparisons of two groups of mice or different primary hepatocytes preparations were carried out using two-tailed unpaired Student's t-test by Graphpad Prism5 (Graphpad Software, San Diego, CA, USA). Differences were considered statistically significant at P<0.05. No statistical methods were used to predetermine sample size. All experiments were performed on at least two independent occasions.

The authors declare that the data supporting the findings of this study are available within the article and itsfiles. Supplementary Information

The authors declare that the data supporting the findings of this study are available within the article and itsfiles. Supplementary Information