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BMAL1-dependent regulation of the mTOR signaling pathway delays aging.

Khapre RV, Kondratova AA, Patel S, Dubrovsky Y, Wrobel M, Antoch MP, Kondratov RV - Aging (Albany NY) (2014)

Bottom Line: Increased mTOR signaling is associated with accelerated aging; in accordance with that, treatment with the mTORC1 inhibitor rapamycin increased lifespan of Bmal1-/- mice by 50%.Our data suggest that BMAL1 is a negative regulator of mTORC1 signaling.We propose that the circadian clock controls the activity of the mTOR pathway through BMAL1-dependent mechanisms and this regulation is important for control of aging and metabolism.

View Article: PubMed Central - PubMed

Affiliation: Center for Gene Regulation in Health and Diseases, BGES, Cleveland State University, Cleveland, OH.

ABSTRACT
The circadian clock, an internal time-keeping system, has been linked with control of aging, but molecular mechanisms of regulation are not known. BMAL1 is a transcriptional factor and core component of the circadian clock; BMAL1 deficiency is associated with premature aging and reduced lifespan. Here we report that activity of mammalian Target of Rapamycin Complex 1 (mTORC1) is increased upon BMAL1 deficiency both in vivo and in cell culture. Increased mTOR signaling is associated with accelerated aging; in accordance with that, treatment with the mTORC1 inhibitor rapamycin increased lifespan of Bmal1-/- mice by 50%. Our data suggest that BMAL1 is a negative regulator of mTORC1 signaling. We propose that the circadian clock controls the activity of the mTOR pathway through BMAL1-dependent mechanisms and this regulation is important for control of aging and metabolism.

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BMAL1 deficiency results in increased mTORC1 signaling in the liver of TRF micePhosphorylation and expression of TORC1 downstream targets were analyzed by western blotting, image analyzing software was used to quantify the intensity of bands. For every experiment the value of maximum intensity of the band for wild type mice was set as 100, the intensity of bands for other time points and for Bmal1−/− mice was normalized accordingly. Results represent average for 4 animals of each genotype for each time point. (a-c) Quantitative profiles of TORC1 downstream targets phosphorylation in the liver of wild type (black diamonds) and Bmal1−/− (grey squares) TRF mice. (a) 4E-BP1 T37/46 phosphorylation. (b) S6K1 T389 phosphorylation; (c) S6K1 T421/424 phosphorylation. * - statistically significant difference between the genotypes. Food was provided at time point 0. Bars on the top of the figure represent light (open bars) and dark (black bars) parts of the day.
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Figure 4: BMAL1 deficiency results in increased mTORC1 signaling in the liver of TRF micePhosphorylation and expression of TORC1 downstream targets were analyzed by western blotting, image analyzing software was used to quantify the intensity of bands. For every experiment the value of maximum intensity of the band for wild type mice was set as 100, the intensity of bands for other time points and for Bmal1−/− mice was normalized accordingly. Results represent average for 4 animals of each genotype for each time point. (a-c) Quantitative profiles of TORC1 downstream targets phosphorylation in the liver of wild type (black diamonds) and Bmal1−/− (grey squares) TRF mice. (a) 4E-BP1 T37/46 phosphorylation. (b) S6K1 T389 phosphorylation; (c) S6K1 T421/424 phosphorylation. * - statistically significant difference between the genotypes. Food was provided at time point 0. Bars on the top of the figure represent light (open bars) and dark (black bars) parts of the day.

Mentions: In order to investigate if BMAL1 is involved in the regulation of mTORC1 activity in vivo, we compared temporal profiles of phosphorylation of the mTORC1 targets in tissues of wild type and Bmal1−/− mice. We assayed the activity of mTORC1 in the liver (Figure 3b), heart (Figure 3d) and several brain regions (cerebellum, Figure 3a and frontal cortex, Figure 3c) isolated from wild type and Bmal1−/− mice across the circadian cycle (on Figure 3 zt0 represents the time when light is on and zt12 represents the time when light is off). In good agreement with in vitro data, we observed a significant increase in phosphorylation of mTORC1 downstream targets at several time points in different tissues of Bmal1−/− mice: in the cerebellum significant increased activity was observed at zt6 (Figure 3a), in the frontal cortex - at zt2 and zt6 (Figure 3c), in the liver - at zt10 and zt18 (Figure 3b) and in the heart - at zt10 and zt14(Figure 3d). The observed difference in mTORC1 signaling between wild type and Bmal1−/− mice can be a consequence of different feeding habits and amount of food consumed by animals: indeed, Bmal1−/− mice do not display circadian rhythms in gene expression and behavior [31], which can affect their feeding. To exclude this factor of variability, we subjected both wild type and Bmal1−/− mice to time-restricted feeding (TRF). Mice of both genotypes received the same amount of food (about 95-100% of their daily intake) at the same time. Both groups consumed the food during first three hours after feeding. As shown in Figure 4, although phosphory-lation of S6K1, 4EBP1 and S6 was induced by feeding and gradually reduced with time in both genotypes, the level of phosphorylation in the liver of Bmal1−/− mice was higher and reduction was considerably delayed compared with wild type. Statistically significant increase in phosphorylation of 4E-BP1 at T37/46 (Figure 4a) and S6K1 at T389 (Figure 4b) was observed at several time points across the daily cycle. This difference cannot arise from variations in the amount of food or feeding behavior, as mice of both genotypes consumed the same amount of food for the same period of time. Taken together, these data suggest that BMAL1 is a negative regulator of mTORC1 signaling in vitro and in vivo. Importantly, BMAL1-dependent regulation was specific for mTORC1-mediated phosphorylation, because we did not detect increased phosphorylation for the MAPK-specific site T421/S424 of S6K1in the Bmal1−/− liver (Figure 4c).


BMAL1-dependent regulation of the mTOR signaling pathway delays aging.

Khapre RV, Kondratova AA, Patel S, Dubrovsky Y, Wrobel M, Antoch MP, Kondratov RV - Aging (Albany NY) (2014)

BMAL1 deficiency results in increased mTORC1 signaling in the liver of TRF micePhosphorylation and expression of TORC1 downstream targets were analyzed by western blotting, image analyzing software was used to quantify the intensity of bands. For every experiment the value of maximum intensity of the band for wild type mice was set as 100, the intensity of bands for other time points and for Bmal1−/− mice was normalized accordingly. Results represent average for 4 animals of each genotype for each time point. (a-c) Quantitative profiles of TORC1 downstream targets phosphorylation in the liver of wild type (black diamonds) and Bmal1−/− (grey squares) TRF mice. (a) 4E-BP1 T37/46 phosphorylation. (b) S6K1 T389 phosphorylation; (c) S6K1 T421/424 phosphorylation. * - statistically significant difference between the genotypes. Food was provided at time point 0. Bars on the top of the figure represent light (open bars) and dark (black bars) parts of the day.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3927809&req=5

Figure 4: BMAL1 deficiency results in increased mTORC1 signaling in the liver of TRF micePhosphorylation and expression of TORC1 downstream targets were analyzed by western blotting, image analyzing software was used to quantify the intensity of bands. For every experiment the value of maximum intensity of the band for wild type mice was set as 100, the intensity of bands for other time points and for Bmal1−/− mice was normalized accordingly. Results represent average for 4 animals of each genotype for each time point. (a-c) Quantitative profiles of TORC1 downstream targets phosphorylation in the liver of wild type (black diamonds) and Bmal1−/− (grey squares) TRF mice. (a) 4E-BP1 T37/46 phosphorylation. (b) S6K1 T389 phosphorylation; (c) S6K1 T421/424 phosphorylation. * - statistically significant difference between the genotypes. Food was provided at time point 0. Bars on the top of the figure represent light (open bars) and dark (black bars) parts of the day.
Mentions: In order to investigate if BMAL1 is involved in the regulation of mTORC1 activity in vivo, we compared temporal profiles of phosphorylation of the mTORC1 targets in tissues of wild type and Bmal1−/− mice. We assayed the activity of mTORC1 in the liver (Figure 3b), heart (Figure 3d) and several brain regions (cerebellum, Figure 3a and frontal cortex, Figure 3c) isolated from wild type and Bmal1−/− mice across the circadian cycle (on Figure 3 zt0 represents the time when light is on and zt12 represents the time when light is off). In good agreement with in vitro data, we observed a significant increase in phosphorylation of mTORC1 downstream targets at several time points in different tissues of Bmal1−/− mice: in the cerebellum significant increased activity was observed at zt6 (Figure 3a), in the frontal cortex - at zt2 and zt6 (Figure 3c), in the liver - at zt10 and zt18 (Figure 3b) and in the heart - at zt10 and zt14(Figure 3d). The observed difference in mTORC1 signaling between wild type and Bmal1−/− mice can be a consequence of different feeding habits and amount of food consumed by animals: indeed, Bmal1−/− mice do not display circadian rhythms in gene expression and behavior [31], which can affect their feeding. To exclude this factor of variability, we subjected both wild type and Bmal1−/− mice to time-restricted feeding (TRF). Mice of both genotypes received the same amount of food (about 95-100% of their daily intake) at the same time. Both groups consumed the food during first three hours after feeding. As shown in Figure 4, although phosphory-lation of S6K1, 4EBP1 and S6 was induced by feeding and gradually reduced with time in both genotypes, the level of phosphorylation in the liver of Bmal1−/− mice was higher and reduction was considerably delayed compared with wild type. Statistically significant increase in phosphorylation of 4E-BP1 at T37/46 (Figure 4a) and S6K1 at T389 (Figure 4b) was observed at several time points across the daily cycle. This difference cannot arise from variations in the amount of food or feeding behavior, as mice of both genotypes consumed the same amount of food for the same period of time. Taken together, these data suggest that BMAL1 is a negative regulator of mTORC1 signaling in vitro and in vivo. Importantly, BMAL1-dependent regulation was specific for mTORC1-mediated phosphorylation, because we did not detect increased phosphorylation for the MAPK-specific site T421/S424 of S6K1in the Bmal1−/− liver (Figure 4c).

Bottom Line: Increased mTOR signaling is associated with accelerated aging; in accordance with that, treatment with the mTORC1 inhibitor rapamycin increased lifespan of Bmal1-/- mice by 50%.Our data suggest that BMAL1 is a negative regulator of mTORC1 signaling.We propose that the circadian clock controls the activity of the mTOR pathway through BMAL1-dependent mechanisms and this regulation is important for control of aging and metabolism.

View Article: PubMed Central - PubMed

Affiliation: Center for Gene Regulation in Health and Diseases, BGES, Cleveland State University, Cleveland, OH.

ABSTRACT
The circadian clock, an internal time-keeping system, has been linked with control of aging, but molecular mechanisms of regulation are not known. BMAL1 is a transcriptional factor and core component of the circadian clock; BMAL1 deficiency is associated with premature aging and reduced lifespan. Here we report that activity of mammalian Target of Rapamycin Complex 1 (mTORC1) is increased upon BMAL1 deficiency both in vivo and in cell culture. Increased mTOR signaling is associated with accelerated aging; in accordance with that, treatment with the mTORC1 inhibitor rapamycin increased lifespan of Bmal1-/- mice by 50%. Our data suggest that BMAL1 is a negative regulator of mTORC1 signaling. We propose that the circadian clock controls the activity of the mTOR pathway through BMAL1-dependent mechanisms and this regulation is important for control of aging and metabolism.

Show MeSH
Related in: MedlinePlus