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• W2066745202 abstract "•A peptide inhibitor of insulin attenuates feeding-induced circadian phase adjustment•Insulin-induced phase shift in peripheral clocks is dependent on tissue type•Transient activation of Per2 is sufficient for phase-dependent circadian shifts•Circadian entrainment in insulin-sensitive tissues involves PI3K and MAPK pathways The circadian clock is entrained to environmental cycles by external cue-mediated phase adjustment. Although the light input pathway has been well defined, the mechanism of feeding-induced phase resetting remains unclear. The tissue-specific sensitivity of peripheral entrainment to feeding suggests the involvement of multiple pathways, including humoral and neuronal signals. Previous in vitro studies with cultured cells indicate that endocrine factors may function as entrainment cues for peripheral clocks. However, blood-borne factors that are well characterized in actual feeding-induced resetting have yet to be identified. Here, we report that insulin may be involved in feeding-induced tissue-type-dependent entrainment in vivo. In ex vivo culture experiments, insulin-induced phase shift in peripheral clocks was dependent on tissue type, which was consistent with tissue-specific insulin sensitivity, and peripheral entrainment in insulin-sensitive tissues involved PI3K- and MAPK-mediated signaling pathways. These results suggest that insulin may be an immediate early factor in feeding-mediated tissue-specific entrainment. The circadian clock is entrained to environmental cycles by external cue-mediated phase adjustment. Although the light input pathway has been well defined, the mechanism of feeding-induced phase resetting remains unclear. The tissue-specific sensitivity of peripheral entrainment to feeding suggests the involvement of multiple pathways, including humoral and neuronal signals. Previous in vitro studies with cultured cells indicate that endocrine factors may function as entrainment cues for peripheral clocks. However, blood-borne factors that are well characterized in actual feeding-induced resetting have yet to be identified. Here, we report that insulin may be involved in feeding-induced tissue-type-dependent entrainment in vivo. In ex vivo culture experiments, insulin-induced phase shift in peripheral clocks was dependent on tissue type, which was consistent with tissue-specific insulin sensitivity, and peripheral entrainment in insulin-sensitive tissues involved PI3K- and MAPK-mediated signaling pathways. These results suggest that insulin may be an immediate early factor in feeding-mediated tissue-specific entrainment. IntroductionThe circadian clock is driven by cell-autonomous clock gene expression rhythms in almost all organisms (Reppert and Weaver, 2002Reppert S.M. Weaver D.R. Coordination of circadian timing in mammals.Nature. 2002; 418: 935-941Crossref PubMed Scopus (3317) Google Scholar, Rosbash et al., 2007Rosbash M. Bradley S. Kadener S. Li Y. Luo W. Menet J.S. Nagoshi E. Palm K. Schoer R. Shang Y. Tang C.H. Transcriptional feedback and definition of the circadian pacemaker in Drosophila and animals.Cold Spring Harb. Symp. Quant. Biol. 2007; 72: 75-83Crossref PubMed Scopus (32) Google Scholar). The core circadian transcriptional feedback loop generates circadian expression of a wide range of numerous genes, which in turn leads to circadian oscillation in diverse physiological processes (Doherty and Kay, 2010Doherty C.J. Kay S.A. Circadian control of global gene expression patterns.Annu. Rev. Genet. 2010; 44: 419-444Crossref PubMed Scopus (210) Google Scholar, Mohawk et al., 2012Mohawk J.A. Green C.B. Takahashi J.S. Central and peripheral circadian clocks in mammals.Annu. Rev. Neurosci. 2012; 35: 445-462Crossref PubMed Scopus (1315) Google Scholar). The circadian clock enables maximum expression of genes at appropriate times of the day, allowing organisms to adapt to earth rotation. It has been reported that chronic desynchronization between physiological and environmental rhythms carries a significant risk of diverse disorders, ranging from sleep disorders to diabetes, cardiovascular diseases, and cancer (Sahar and Sassone-Corsi, 2012Sahar S. Sassone-Corsi P. Regulation of metabolism: the circadian clock dictates the time.Trends Endocrinol. Metab. 2012; 23: 1-8Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, Wijnen and Young, 2006Wijnen H. Young M.W. Interplay of circadian clocks and metabolic rhythms.Annu. Rev. Genet. 2006; 40: 409-448Crossref PubMed Scopus (277) Google Scholar). Thus, a thorough understanding of the mechanism of the circadian input pathway is critically important to the prevention of diseases. The circadian input consists of two major pathways. The first is the light input pathway via the hypothalamic suprachiasmatic nuclei (SCN), known as the circadian pacemaker. Details of this mechanism have been elucidated at the molecular level by a substantial number of studies (Doyle and Menaker, 2007Doyle S. Menaker M. Circadian photoreception in vertebrates.Cold Spring Harb. Symp. Quant. Biol. 2007; 72: 499-508Crossref PubMed Scopus (47) Google Scholar). The second is the feeding input pathway (Damiola et al., 2000Damiola F. Le Minh N. Preitner N. Kornmann B. Fleury-Olela F. Schibler U. Restricted feeding uncouples circadian oscillators in peripheral tissues from the central pacemaker in the suprachiasmatic nucleus.Genes Dev. 2000; 14: 2950-2961Crossref PubMed Scopus (1714) Google Scholar, Stokkan et al., 2001Stokkan K.A. Yamazaki S. Tei H. Sakaki Y. Menaker M. Entrainment of the circadian clock in the liver by feeding.Science. 2001; 291: 490-493Crossref PubMed Scopus (1341) Google Scholar), for which a factor has yet to be identified and characterized in vivo. Temporal feeding restriction changes the phase of circadian gene expression in peripheral tissues without affecting the phase in the SCN, and food-induced phase resetting proceeds much faster in the liver than in the kidney, heart, or pancreas (Damiola et al., 2000Damiola F. Le Minh N. Preitner N. Kornmann B. Fleury-Olela F. Schibler U. Restricted feeding uncouples circadian oscillators in peripheral tissues from the central pacemaker in the suprachiasmatic nucleus.Genes Dev. 2000; 14: 2950-2961Crossref PubMed Scopus (1714) Google Scholar). The tissue-specific sensitivity of peripheral entrainment to feeding suggests the involvement of multiple pathways, including humoral and neuronal signals.The circadian clock is cell-autonomous (Nagoshi et al., 2004Nagoshi E. Saini C. Bauer C. Laroche T. Naef F. Schibler U. Circadian gene expression in individual fibroblasts: cell-autonomous and self-sustained oscillators pass time to daughter cells.Cell. 2004; 119: 693-705Abstract Full Text Full Text PDF PubMed Scopus (771) Google Scholar, Yamazaki et al., 2000Yamazaki S. Numano R. Abe M. Hida A. Takahashi R. Ueda M. Block G.D. Sakaki Y. Menaker M. Tei H. Resetting central and peripheral circadian oscillators in transgenic rats.Science. 2000; 288: 682-685Crossref PubMed Scopus (1478) Google Scholar), and much can therefore be learned from studies using cultured cells. Indeed, cell line-based experiments to elucidate the entrainment mechanism have been performed. A number of endogenous factors are reported to act on cell-autonomous circadian gene expression in cultured cell lines, including growth factors, calcium, glucose, angiotensin II, retinoic acid, and nitric oxide (Akashi and Nishida, 2000Akashi M. Nishida E. Involvement of the MAP kinase cascade in resetting of the mammalian circadian clock.Genes Dev. 2000; 14: 645-649PubMed Google Scholar, Balsalobre et al., 2000Balsalobre A. Marcacci L. Schibler U. Multiple signaling pathways elicit circadian gene expression in cultured Rat-1 fibroblasts.Curr. Biol. 2000; 10: 1291-1294Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar, Hirota et al., 2002Hirota T. Okano T. Kokame K. Shirotani-Ikejima H. Miyata T. Fukada Y. Glucose down-regulates Per1 and Per2 mRNA levels and induces circadian gene expression in cultured Rat-1 fibroblasts.J. Biol. Chem. 2002; 277: 44244-44251Crossref PubMed Scopus (259) Google Scholar, McNamara et al., 2001McNamara P. Seo S.B. Rudic R.D. Sehgal A. Chakravarti D. FitzGerald G.A. Regulation of CLOCK and MOP4 by nuclear hormone receptors in the vasculature: a humoral mechanism to reset a peripheral clock.Cell. 2001; 105: 877-889Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar, Nonaka et al., 2001Nonaka H. Emoto N. Ikeda K. Fukuya H. Rohman M.S. Raharjo S.B. Yagita K. Okamura H. Yokoyama M. Angiotensin II induces circadian gene expression of clock genes in cultured vascular smooth muscle cells.Circulation. 2001; 104: 1746-1748Crossref PubMed Scopus (157) Google Scholar). In addition, insulin, a humoral factor that regulates blood glucose levels, also has effects on clock gene expression in cultured cell lines (Balsalobre et al., 2000Balsalobre A. Marcacci L. Schibler U. Multiple signaling pathways elicit circadian gene expression in cultured Rat-1 fibroblasts.Curr. Biol. 2000; 10: 1291-1294Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar, Yamajuku et al., 2012Yamajuku D. Inagaki T. Haruma T. Okubo S. Kataoka Y. Kobayashi S. Ikegami K. Laurent T. Kojima T. Noutomi K. et al.Real-time monitoring in three-dimensional hepatocytes reveals that insulin acts as a synchronizer for liver clock.Sci Rep. 2012; 2: 439Crossref PubMed Scopus (86) Google Scholar). With regard to in vivo relevance, mice carrying pharmacologically damaged beta cells show altered expression of clock genes in peripheral tissues (Kuriyama et al., 2004Kuriyama K. Sasahara K. Kudo T. Shibata S. Daily injection of insulin attenuated impairment of liver circadian clock oscillation in the streptozotocin-treated diabetic mouse.FEBS Lett. 2004; 572: 206-210Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, Oishi et al., 2004Oishi K. Kasamatsu M. Ishida N. Gene- and tissue-specific alterations of circadian clock gene expression in streptozotocin-induced diabetic mice under restricted feeding.Biochem. Biophys. Res. Commun. 2004; 317: 330-334Crossref PubMed Scopus (96) Google Scholar), indicating the presence of in vivo interaction between the circadian clock and insulin signaling. However, the physiological role of insulin in the circadian clock remains to be determined.The pancreas secretes insulin in response to feeding. We speculated that insulin acts as an endogenous molecule required for feeding-induced tissue-specific phase resetting of peripheral clocks. To help elucidate the in vivo roles of insulin in feeding-induced circadian entrainment, we used in vivo imaging experiments to examine expression levels of Per2 in individual animals around the clock in the presence of a highly specific competitive peptide inhibitor of insulin. To exclude the possibility that the results were affected by secondary effects of the inhibitor, we used ex vivo approaches. We therefore conducted ex vivo tissue culture experiments to examine whether insulin-induced phase shift in peripheral clocks depends on tissue type, consistent with the tissue-specific sensitivity of insulin.Results and DiscussionEvaluation of peripheral clocks requires the preparation of a large number of animals, harvesting of tissues every few hours, and examination of clock gene expression levels using purified RNA. These experimental procedures are arduous, but provide no information about sequential changes in clock gene expression in individual animals. Rather, the data only show average expression levels from several dead animals. In contrast, a recent in vivo imaging technique enables detection of sequential changes in clock gene expression levels in individual animals without killing them (Tahara et al., 2012Tahara Y. Kuroda H. Saito K. Nakajima Y. Kubo Y. Ohnishi N. Seo Y. Otsuka M. Fuse Y. Ohura Y. et al.In vivo monitoring of peripheral circadian clocks in the mouse.Curr. Biol. 2012; 22: 1029-1034Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Hence, using in vivo imaging of individual Per2-luciferase knockin mice (Yoo et al., 2004Yoo S.H. Yamazaki S. Lowrey P.L. Shimomura K. Ko C.H. Buhr E.D. Siepka S.M. Hong H.K. Oh W.J. Yoo O.J. et al.PERIOD2:LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues.Proc. Natl. Acad. Sci. USA. 2004; 101: 5339-5346Crossref PubMed Scopus (1711) Google Scholar), we investigated temporal changes in Per2 expression rhythms in peripheral tissues of individual animals (Figure 1A, top). About 10 min after subcutaneous injection of luciferin, strong luminescence was detected in the liver and submandibular gland. We were then able to confirm circadian gene expression of Per2 in these tissues by time course measurement around the clock. Furthermore, to observe feeding-induced phase shift of circadian gene expression, we changed the feeding schedule from ZT12-24 to ZT0-6, and 5 days later observed antiphase expression of Per2 in the liver (Figure 1A, right). In contrast, this phase shift was subtle in the submandibular gland (Figures 1A and 1B), suggesting that there are significant differences among peripheral tissues in the rate of feeding-induced phase shift. The phase change in individual animals is shown in Figure 1C. While Per2 expression in each animal’s liver showed a similar and reproducible response to the restricted feeding, minor variation among individuals was apparent in the rate of the phase shift. The pancreas secretes insulin in response to feeding, and peripheral tissues show different sensitivity to insulin, indicating why the liver clock, but not the submandibular gland clock, was immediately phase-adjusted by restricted feeding. These results demonstrate that in vivo imaging technology is a powerful tool in investigating sequential and temporal changes in peripheral clocks in individuals.It was reported that restricted feeding triggers a rapid transient induction of Per2 transcription in the liver (Wu et al., 2010Wu T. Ni Y. Kato H. Fu Z. Feeding-induced rapid resetting of the hepatic circadian clock is associated with acute induction of Per2 and Dec1 transcription in rats.Chronobiol. Int. 2010; 27: 1-18Crossref PubMed Scopus (33) Google Scholar). The immediate early expression of Per2 would likely affect the negative feedback loop in circadian transcription because Per2 is a component of the negative limb. If insulin is indispensable to feeding-induced resetting, inhibition of insulin signaling should result in attenuation of the Per2 expression levels rapidly induced by restricted feeding (Figure 2A). To inhibit insulin action during feeding-induced phase shift without pharmacologically killing beta cells, mice received a subcutaneous injection of S961, a highly specific competitive peptide inhibitor of insulin, prior to feeding (Schäffer et al., 2008Schäffer L. Brand C.L. Hansen B.F. Ribel U. Shaw A.C. Slaaby R. Sturis J. A novel high-affinity peptide antagonist to the insulin receptor.Biochem. Biophys. Res. Commun. 2008; 376: 380-383Crossref PubMed Scopus (112) Google Scholar, Scherer et al., 2011Scherer T. O’Hare J. Diggs-Andrews K. Schweiger M. Cheng B. Lindtner C. Zielinski E. Vempati P. Su K. Dighe S. et al.Brain insulin controls adipose tissue lipolysis and lipogenesis.Cell Metab. 2011; 13: 183-194Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, Vikram and Jena, 2010Vikram A. Jena G. S961, an insulin receptor antagonist causes hyperinsulinemia, insulin-resistance and depletion of energy stores in rats.Biochem. Biophys. Res. Commun. 2010; 398: 260-265Crossref PubMed Scopus (58) Google Scholar). S961-injected animals ate a similar amount of food as control animals. As expected, this pretreatment with S961 potently inhibited the rapid transient induction of Per2 by restricted feeding. Next, using an in vivo imaging system, we examined whether S961 suppresses the feeding-induced circadian phase shift of Per2 expression rhythm in the liver of individual animals (Figure 2B). In the control animals, transition states of Per2 expression rhythms were confirmed in each animal’s liver approximately 2 days after the inversion of feeding time. During the process of transition, individual differences in phase change were observed: some animals showed lower amplitudes of oscillation temporarily, whereas others had a second peak (Figure S1). Average transition states of eight animals are shown in Figure 2C. Compared with the control animals, animals pretreated with S961 showed a significant delay in phase shift on day 2, and phase adjustment was still not completed on day 4 (Figures 2B–2D). When we processed data from individual animals using a cosine curve fitting and calculated and quantified peak times (Figure 2D), we found that the shift speed of acrophase appeared to be attenuated in animals pretreated with S961.Figure 2Involvement of Insulin in Feeding-Induced Circadian Phase Shift in the LiverShow full caption(A) Acute induction of Per2 transcripts after feeding was blocked by S961. Per2 mRNA expression levels in liver at ZT2 (60 min after feeding onset) were determined with RT-PCR. Each value was normalized with β-actin. The data represent the mean ± SE (n = 3). ∗p < 0.05.(B) Representative in vivo image of Per2Luc mice liver injected with S961 30 min before the start of feeding every day during restricted feeding. S961 inhibited the entrainment of Per2.(C) Quantitative data of bioluminescence in the liver from (B). The data represent the mean ± SE (n = 8). ∗∗p < 0.01.(D) Acrophases in a representative animal from (B) were calculated by a cosine curve fitting. The data at right represent the mean ± SE (n = 8).(E) Quantitative RT-PCR of Per2, Bmal1, and Rev-erbα mRNA in the liver during RF (ZT1-7) in LD; n = 2. Black circles represent individual values of mice given PBS mice and green circles represent mice given S961. S961 was used at a concentration of 30 nmol/kg in all experiments.View Large Image Figure ViewerDownload (PPT)A limitation of the present in vivo imaging approach is that it provides no information on expression levels of genes other than Per2. We therefore harvested the livers every 4 hours in the conventional manner and confirmed the effect of S961 on feeding-induced phase shift of clock gene oscillations with RT-PCR (Figure 2E). As is the case with the in vivo imaging data, circadian phase shift of the Per2, Bmal1, and Rev-erbα genes was significantly delayed compared with control animals. Sampling in this experiment was performed under light-dark conditions, but Per2 expression results were similar to the in vivo imaging data obtained under dark-dark conditions (Figure 2B). Furthermore, the S961-treated mice weighed almost the same as the control mice throughout the experiment (Figure S2A). Because S961 treatment provoked transient hyperglycemia (Figure S2B), we used an ex vivo slice culture system to confirm that this hyperglycemia had no substantial influence on the effect of S961 on insulin-induced circadian phase shift (Figure S3).These data illustrate that the inhibition of insulin signaling attenuates feeding-induced phase adjustment of the liver clock in individual animals. As mentioned above, S961 potently inhibited the transient induction of Per2 that was triggered by restricted feeding, whereas the phase shift of Per2 expression rhythms was less strongly affected. This suggests that the transient induction may contribute to the phase shift, but is not absolutely required for it, and that other unknown endogenous factors are involved in the process.There is no doubt that S961 treatment is a superior experimental approach for investigating the physiological role of insulin compared with methods using streptozotocin-treated, ob/ob or db/db mice. However, it is impossible to completely exclude the possibility that various secondary physiological events caused by S961-induced transient hyperglycemia may affect feeding-induced circadian phase shift. To exclude these secondary effects and examine the true contribution of insulin to the circadian phase shift, ex vivo or in vitro experimental approaches are required for further validation. We therefore conducted ex vivo culture experiments to investigate the effect of insulin on autonomous circadian gene expression. Per2 expression rhythms in the liver were phase-shifted by the administration of insulin, and the effect was then almost completely inhibited in the presence of S961, suggesting that insulin acted on the liver clock in a receptor-specific manner (Figure 3A). Additionally, given the well-known decrease in insulin sensitivity in the fatty liver, we examined the effect of insulin on Per2 expression rhythm in livers harvested from mice fed a high-fat diet (Figure 3B). As expected, the insulin-induced phase shift in circadian gene expression was smaller in fatty livers than in healthy livers. We found a phase dependency in the insulin-mediated phase change of Per2 expression rhythms in the liver (Figure 3C). Insulin caused a phase advance effect during the increasing phase of Per2 expression, but a phase delay effect during the decreasing phase. Unexpectedly, the phase responsiveness observed on continuous administration of insulin was similar to that with 1 hr treatment with insulin (Figure 3C, right versus left), indicating the presence of a negative feedback mechanism for blocking continuous activation of the signaling pathway leading to the core clock. We constructed a dot-plot of phase responsiveness on administration of insulin at various circadian phases (Figure 3D). The data indicate that the direction of phase change of peripheral clocks depends on the feeding time of day. To confirm whether the phase shift by insulin is consistent with this tissue insulin sensitivity, we examined the tissue-specific effect of insulin on autonomous Per2 expression rhythms ex vivo by performing slice culture of various peripheral tissues (Figure 3E). As expected, we confirmed a large phase shift of Per2 oscillation not only in liver, but also in adipose tissue, both of which are insulin-sensitive. In contrast, insulin exerted no or only a subtle effect in insensitive tissues, including the lung, aorta, and submandibular gland.Figure 3Insulin-Mediated Circadian Phase Shift of Per2 Expression Rhythms in Insulin-Sensitive TissuesShow full caption(A–E) Explants derived from Per2Luc mice were treated with insulin at specific phases. Bioluminescence was measured in real time.(A) Representative data of liver explants pretreated with 200 nM S961 from 30 min before administration of insulin. Gray shadows represent the presence of insulin and S961 in culture media.(B) Oil Red O staining and representative data of explants derived from high-fat diet (HFD)-fed Per2Luc mice. Gray shadows represent the presence of insulin in culture media. CV, central vein; NC, normal control.(C) Phase-dependent alteration of mice liver explants with insulin. Experiments with a transient treatment (1 hr) of insulin are shown on the left, and experiments with a continuous treatment at right. Gray shadows represent the presence of insulin in culture media. Representative data are shown.(D) Phase response plot of time interval between peaks in liver from (C).(E) Representative data and phase response plot of time interval between peaks of insulin-administered submandibular gland (Sub Gla), lung, aorta, and white adipose tissue (WAT). All explants were treated with dexamethasone (Dex) and the second peak after Dex treatment was defined as peak2 (∗), and the next peak as peak3. Peak2-time was referred to as time = 0. Peak interval was calculated from the time difference between peak4 and peak2. Arrowheads indicate the time of administration. For the analysis of slices other than WAT, data were detrended by subtracting a 24 hr running average from raw data.View Large Image Figure ViewerDownload (PPT)On the assumption that the insulin-induced phase shift is in fact dependent on insulin receptor expression, we speculated that ectopic overexpression of the insulin receptor may confer a degree of insulin sensitivity to receptor-poor cells. To test this notion, we introduced an insulin receptor expression plasmid vector into NIH 3T3 fibroblasts, a receptor-poor cell line (Figure 4A). Insulin receptor overexpression was constitutively driven by the AG promoter. In cells transfected with the empty vector, insulin did not trigger a phase shift in Bmal1 expression rhythm (here using a Bmal1 promoter-driven luciferase expression vector), but did induce a phase shift in cells ectopically overexpressing the insulin receptor. Interestingly, we found a phase difference in insulin sensitivity in spite of constitutive overexpression of the receptor; insulin administration caused a phase advance during the decreasing phase of Bmal1 (the increasing phase of Per2), as shown in the liver, versus a phase delay in the increasing phase (Figure 4B). This may indicate that phase-dependent insulin action might be explained in terms of the internal clock, independently of diurnal changes in receptor expression levels. On the other hand, we found that mRNA levels of the liver insulin receptor showed a clear circadian pattern with an opposite phase to Per2 (Figure S4A), as indicated previously, which might have contributed not to the direction but rather to the intensity of the insulin-induced phase shift.Figure 4Acquisition of Insulin Sensitivity by Ectopic Receptor Expression and Phase-Dependent Circadian Phase Shift by Transient Induction of Per2Show full caption(A) NIH 3T3 fibroblasts were transfected with the Bmal1-driven luciferase and human insulin receptor α (hIRα) subunit expression vectors, and then stimulated with 60 nM insulin or vehicle. Arrowheads indicate the time of administration. To synchronize cellular clocks, the cells were treated with 50 nM dexamethasone more than 24 hr before insulin stimulation.(B) Phase differences were calculated by comparing the first peak or trough after administration of insulin. DL, delay phase administration (increasing phase of Bmal1 expression); ADV, advance phase administration (decreasing phase of Bmal1 expression). Data are represented as the mean ± SE for triplicate samples.(C and D) NIH 3T3 and U2OS cells were transfected with the Bmal1-driven luciferase vector and a Lac repressor-expressing vector in the presence or absence of an IPTG-inducible Per2 expression vector. The cells were treated with 50 nM dexamethasone (2 hr) to synchronize cellular clocks. Bmal1 transcription was monitored in real-time using a cell culture-based luminescent monitoring system in the presence of luciferin. Approximately 18–20 hr (decreasing phase of Bmal1 expression, for “phase advance” experiments) or 30–32 hr (increasing phase of Bmal1 expression, for “phase delay” experiments) after the dexamethasone shock, 2 mM IPTG was added to the culture media to induce Per2 for 1 hr. Arrows indicate the time of administration. One hour after the administration, IPTG was removed.(E and F) Phase differences were calculated by comparing the first peak or trough after administration of IPTG. Data are represented as the mean ± SE for triplicate samples. DL, delay phase administration (increasing phase of Bmal1 expression); ADV, advance phase administration (decreasing phase of Bmal1 expression).(G and H) Liver and white adipose tissue (WAT) explants from Per2Luc mice were pretreated with 50 μM LY294002 (LY) or 20 μM U0126 (U) before administration of insulin. Explants were incubated with insulin for 2 hr. The data represent the mean ± SE. Gray shadows represent the presence of insulin and inhibitors in culture media. For synchronization of cellular clocks, all explants were treated with 100 nM dexamethasone (Dex). The second peak after Dex treatment was defined as peak2 (∗) and the next peak as peak3. Peak2-time was referred to as time = 0. Peak interval was calculated from the time difference between peak3 and peak2. Arrowheads indicate the time of administration. For the analysis of slices other than WAT, data were detrended by subtracting a 24 hr running average from raw data.View Large Image Figure ViewerDownload (PPT)To examine whether transient activation of Per2 transcription is sufficient to induce circadian phase shift in a phase-directed manner, we introduced an isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible expression system to two cell lines, NIH 3T3 and U2OS (Figures 4C and 4D). In this culture system, whereas ectopic expression of Per2 is suppressed by a lac repressor in the absence of IPTG, it can be rapidly activated by administration of IPTG without extracellular physiological stimuli and independently of any intracellular signaling pathways. A 1-hr administration of IPTG caused a 1.5-hr phase delay during the increasing phase of Bmal1 (decreasing phase of Per2), versus a 1.5-hr phase advance in the decreasing phase, in both the cell lines (Figures 4E and 4F). Although the magnitude of phase shifts was not particularly large when compared with the insulin-induced phase shift in the liver, the direction of the phase shifts by insulin treatment was reproduced by simply inducing ectopic expression of Per2. These results support the possibility that the phase responsiveness is explained in terms of the internal clock, independently of any extracellular stimuli or intracellular signaling pathways.Activation of the insulin receptor leads to cellular glucose uptake through the glucose transporter GLUT4, and activation of the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MA" @default.
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• W2066745202 title "The Role of the Endocrine System in Feeding-Induced Tissue-Specific Circadian Entrainment" @default.
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