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Nocturnin expression is induced by fasting in the white adipose tissue of restricted fed mice.

Gilbert MR, Douris N, Tongjai S, Green CB - PLoS ONE (2011)

Bottom Line: The relationship between circadian clocks and metabolism is intimate and complex and a number of recent studies have begun to reveal previously unknown effects of food and its temporal availability on the clock and the rhythmic transcriptome of peripheral tissues.A rise in cAMP levels also induces Nocturnin expression, suggesting that Nocturnin's induction in eWAT by fasting is likely mediated through the same pathways that activate lipolysis.Therefore, this suggests that Nocturnin plays a role in linking nutrient sensing by the circadian clock to lipid mobilization in the adipocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Virginia, Charlottesville, Virginia, United States of America.

ABSTRACT
The relationship between circadian clocks and metabolism is intimate and complex and a number of recent studies have begun to reveal previously unknown effects of food and its temporal availability on the clock and the rhythmic transcriptome of peripheral tissues. Nocturnin, a circadian deadenylase, is expressed rhythmically in a wide variety of tissues, but we report here that Nocturnin expression is arrhythmic in epididymal white adipose tissue (eWAT) of mice housed in 12:12 LD with ad libitum access to food. However, Nocturnin expression becomes rhythmic in eWAT of mice placed on restricted feeding. We show here that Nocturnin's rhythmic expression pattern is not dependent upon feeding, nor is it acutely induced by feeding in the liver or eWAT of ad libitum fed mice. However, Nocturnin is acutely induced by the absence of the expected meal in eWAT of restricted fed mice. A rise in cAMP levels also induces Nocturnin expression, suggesting that Nocturnin's induction in eWAT by fasting is likely mediated through the same pathways that activate lipolysis. Therefore, this suggests that Nocturnin plays a role in linking nutrient sensing by the circadian clock to lipid mobilization in the adipocytes.

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Related in: MedlinePlus

Nocturnin is induced by a rise in cAMP in NIH-3T3 fibroblasts and in 3T3-L1 adipocytes.(A) Schematic representation of highly conserved putative transcription factor binding sites in the 10 kb region upstream of Nocturnin's translation start site. C =  cAMP response element binding protein (CRE) sites. E =  E-box elements. Locations of the CRE sites are C1 −29 bp and C2+3,016 bp. (B) Nocturnin is induced approximately 4.5 fold by IBMX (solid line) in NIH3T3 fibroblasts. No induction of Nocturnin was observed in the vehicle treated cells (dashed line). Data represent mean (± SEM), normalized to Cyclophilin B. n = 4 for each time point. P-value determined by t-test assuming equal variances, two-way p-value reported. ** p-value <0.002; * p-value <0.03. (C) Nocturnin is induced approximately 2.5 fold by forskolin (solid line) in NIH3T3 fibroblasts. The dashed line represents data from vehicle treated cells. Data represent mean (± SEM), normalized to Cyclophilin B. n = 3 for each time point. P-value determined by t-test assuming equal variances, two-way p-value reported. * p-value <0.03. (D) Transient transfection assay of NIH3T3 fibroblasts showing Nocturnin is induced by the overexpression of PKA in a CREB-dependent manner. Data represent the mean (± SEM), n = 6 for each group. T-test assuming equal variances performed, p-value reported. **  =  p-value <0.01; *  =  p-value <0.04. NT =  non-transfected; GFP =  GFP transfected; K-CREB =  pCMV-KCREB (dominant negative) alone transfected; PKA =  pCMV-PKA transfected; PKA + K-CREB  =  pCMV-PKA and dominant negative pCMV-KCREB were cotransfected. (E) Nocturnin is induced approximately 2 fold by forskolin (solid line) in mature 3T3-L1 adipocytes. The dashed line represents data from vehicle treated cells. Data represent the mean (± SEM), n = 3. **  =  p-value 0.0009, and *  =  p-value 0.036. Data were normalized to Cyclophilin B.
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pone-0017051-g005: Nocturnin is induced by a rise in cAMP in NIH-3T3 fibroblasts and in 3T3-L1 adipocytes.(A) Schematic representation of highly conserved putative transcription factor binding sites in the 10 kb region upstream of Nocturnin's translation start site. C =  cAMP response element binding protein (CRE) sites. E =  E-box elements. Locations of the CRE sites are C1 −29 bp and C2+3,016 bp. (B) Nocturnin is induced approximately 4.5 fold by IBMX (solid line) in NIH3T3 fibroblasts. No induction of Nocturnin was observed in the vehicle treated cells (dashed line). Data represent mean (± SEM), normalized to Cyclophilin B. n = 4 for each time point. P-value determined by t-test assuming equal variances, two-way p-value reported. ** p-value <0.002; * p-value <0.03. (C) Nocturnin is induced approximately 2.5 fold by forskolin (solid line) in NIH3T3 fibroblasts. The dashed line represents data from vehicle treated cells. Data represent mean (± SEM), normalized to Cyclophilin B. n = 3 for each time point. P-value determined by t-test assuming equal variances, two-way p-value reported. * p-value <0.03. (D) Transient transfection assay of NIH3T3 fibroblasts showing Nocturnin is induced by the overexpression of PKA in a CREB-dependent manner. Data represent the mean (± SEM), n = 6 for each group. T-test assuming equal variances performed, p-value reported. **  =  p-value <0.01; *  =  p-value <0.04. NT =  non-transfected; GFP =  GFP transfected; K-CREB =  pCMV-KCREB (dominant negative) alone transfected; PKA =  pCMV-PKA transfected; PKA + K-CREB  =  pCMV-PKA and dominant negative pCMV-KCREB were cotransfected. (E) Nocturnin is induced approximately 2 fold by forskolin (solid line) in mature 3T3-L1 adipocytes. The dashed line represents data from vehicle treated cells. Data represent the mean (± SEM), n = 3. **  =  p-value 0.0009, and *  =  p-value 0.036. Data were normalized to Cyclophilin B.

Mentions: During fasting, white adipose tissue undergoes lipolysis mediated by catecholamine stimulated beta-adrenergic receptors which causes a rise in cAMP levels, leading to the activation of protein kinase A (PKA) which results in the phosphorylation of hormone sensitive lipase (HSL), perilipin A, and cAMP response element binding protein (CREB), and ultimately the breakdown of triglyceride-storing lipid droplets [24], [25], [26], [27]. It has been shown in Xenopus laevis that Nocturnin rhythmicity is regulated by p-CREB, but the activation of Nocturnin by p-CREB has not been demonstrated in mice [28]. There are conserved putative CREs within the 500 bp region upstream of Nocturnin's first exon (Figure 5A), and Nocturnin is acutely induced by 3-isobutyl-1-methylxanthine (IBMX) and forskolin in NIH3T3 fibroblasts (Figure 5B,C). Overexpression of PKA in NIH-3T3 fibroblasts resulted in an increase of Nocturnin expression, whereas cells co-transfected with PKA and a dominant negative CREB construct failed to exhibit the increase in Nocturnin expression, demonstrating that mammalian Nocturnin mRNA expression is induced by CREB activation (Figure 5D). We next examined whether Nocturnin could be induced by a rise in cAMP levels in mature 3T3-L1 adipocytes. Treatment of these cells with forskolin also resulted in an acute induction of Nocturnin expression (Figure 5E) showing that Nocturnin is regulated by the signaling cascade which occurs during lipolysis in white adipose tissue.


Nocturnin expression is induced by fasting in the white adipose tissue of restricted fed mice.

Gilbert MR, Douris N, Tongjai S, Green CB - PLoS ONE (2011)

Nocturnin is induced by a rise in cAMP in NIH-3T3 fibroblasts and in 3T3-L1 adipocytes.(A) Schematic representation of highly conserved putative transcription factor binding sites in the 10 kb region upstream of Nocturnin's translation start site. C =  cAMP response element binding protein (CRE) sites. E =  E-box elements. Locations of the CRE sites are C1 −29 bp and C2+3,016 bp. (B) Nocturnin is induced approximately 4.5 fold by IBMX (solid line) in NIH3T3 fibroblasts. No induction of Nocturnin was observed in the vehicle treated cells (dashed line). Data represent mean (± SEM), normalized to Cyclophilin B. n = 4 for each time point. P-value determined by t-test assuming equal variances, two-way p-value reported. ** p-value <0.002; * p-value <0.03. (C) Nocturnin is induced approximately 2.5 fold by forskolin (solid line) in NIH3T3 fibroblasts. The dashed line represents data from vehicle treated cells. Data represent mean (± SEM), normalized to Cyclophilin B. n = 3 for each time point. P-value determined by t-test assuming equal variances, two-way p-value reported. * p-value <0.03. (D) Transient transfection assay of NIH3T3 fibroblasts showing Nocturnin is induced by the overexpression of PKA in a CREB-dependent manner. Data represent the mean (± SEM), n = 6 for each group. T-test assuming equal variances performed, p-value reported. **  =  p-value <0.01; *  =  p-value <0.04. NT =  non-transfected; GFP =  GFP transfected; K-CREB =  pCMV-KCREB (dominant negative) alone transfected; PKA =  pCMV-PKA transfected; PKA + K-CREB  =  pCMV-PKA and dominant negative pCMV-KCREB were cotransfected. (E) Nocturnin is induced approximately 2 fold by forskolin (solid line) in mature 3T3-L1 adipocytes. The dashed line represents data from vehicle treated cells. Data represent the mean (± SEM), n = 3. **  =  p-value 0.0009, and *  =  p-value 0.036. Data were normalized to Cyclophilin B.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3037405&req=5

pone-0017051-g005: Nocturnin is induced by a rise in cAMP in NIH-3T3 fibroblasts and in 3T3-L1 adipocytes.(A) Schematic representation of highly conserved putative transcription factor binding sites in the 10 kb region upstream of Nocturnin's translation start site. C =  cAMP response element binding protein (CRE) sites. E =  E-box elements. Locations of the CRE sites are C1 −29 bp and C2+3,016 bp. (B) Nocturnin is induced approximately 4.5 fold by IBMX (solid line) in NIH3T3 fibroblasts. No induction of Nocturnin was observed in the vehicle treated cells (dashed line). Data represent mean (± SEM), normalized to Cyclophilin B. n = 4 for each time point. P-value determined by t-test assuming equal variances, two-way p-value reported. ** p-value <0.002; * p-value <0.03. (C) Nocturnin is induced approximately 2.5 fold by forskolin (solid line) in NIH3T3 fibroblasts. The dashed line represents data from vehicle treated cells. Data represent mean (± SEM), normalized to Cyclophilin B. n = 3 for each time point. P-value determined by t-test assuming equal variances, two-way p-value reported. * p-value <0.03. (D) Transient transfection assay of NIH3T3 fibroblasts showing Nocturnin is induced by the overexpression of PKA in a CREB-dependent manner. Data represent the mean (± SEM), n = 6 for each group. T-test assuming equal variances performed, p-value reported. **  =  p-value <0.01; *  =  p-value <0.04. NT =  non-transfected; GFP =  GFP transfected; K-CREB =  pCMV-KCREB (dominant negative) alone transfected; PKA =  pCMV-PKA transfected; PKA + K-CREB  =  pCMV-PKA and dominant negative pCMV-KCREB were cotransfected. (E) Nocturnin is induced approximately 2 fold by forskolin (solid line) in mature 3T3-L1 adipocytes. The dashed line represents data from vehicle treated cells. Data represent the mean (± SEM), n = 3. **  =  p-value 0.0009, and *  =  p-value 0.036. Data were normalized to Cyclophilin B.
Mentions: During fasting, white adipose tissue undergoes lipolysis mediated by catecholamine stimulated beta-adrenergic receptors which causes a rise in cAMP levels, leading to the activation of protein kinase A (PKA) which results in the phosphorylation of hormone sensitive lipase (HSL), perilipin A, and cAMP response element binding protein (CREB), and ultimately the breakdown of triglyceride-storing lipid droplets [24], [25], [26], [27]. It has been shown in Xenopus laevis that Nocturnin rhythmicity is regulated by p-CREB, but the activation of Nocturnin by p-CREB has not been demonstrated in mice [28]. There are conserved putative CREs within the 500 bp region upstream of Nocturnin's first exon (Figure 5A), and Nocturnin is acutely induced by 3-isobutyl-1-methylxanthine (IBMX) and forskolin in NIH3T3 fibroblasts (Figure 5B,C). Overexpression of PKA in NIH-3T3 fibroblasts resulted in an increase of Nocturnin expression, whereas cells co-transfected with PKA and a dominant negative CREB construct failed to exhibit the increase in Nocturnin expression, demonstrating that mammalian Nocturnin mRNA expression is induced by CREB activation (Figure 5D). We next examined whether Nocturnin could be induced by a rise in cAMP levels in mature 3T3-L1 adipocytes. Treatment of these cells with forskolin also resulted in an acute induction of Nocturnin expression (Figure 5E) showing that Nocturnin is regulated by the signaling cascade which occurs during lipolysis in white adipose tissue.

Bottom Line: The relationship between circadian clocks and metabolism is intimate and complex and a number of recent studies have begun to reveal previously unknown effects of food and its temporal availability on the clock and the rhythmic transcriptome of peripheral tissues.A rise in cAMP levels also induces Nocturnin expression, suggesting that Nocturnin's induction in eWAT by fasting is likely mediated through the same pathways that activate lipolysis.Therefore, this suggests that Nocturnin plays a role in linking nutrient sensing by the circadian clock to lipid mobilization in the adipocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Virginia, Charlottesville, Virginia, United States of America.

ABSTRACT
The relationship between circadian clocks and metabolism is intimate and complex and a number of recent studies have begun to reveal previously unknown effects of food and its temporal availability on the clock and the rhythmic transcriptome of peripheral tissues. Nocturnin, a circadian deadenylase, is expressed rhythmically in a wide variety of tissues, but we report here that Nocturnin expression is arrhythmic in epididymal white adipose tissue (eWAT) of mice housed in 12:12 LD with ad libitum access to food. However, Nocturnin expression becomes rhythmic in eWAT of mice placed on restricted feeding. We show here that Nocturnin's rhythmic expression pattern is not dependent upon feeding, nor is it acutely induced by feeding in the liver or eWAT of ad libitum fed mice. However, Nocturnin is acutely induced by the absence of the expected meal in eWAT of restricted fed mice. A rise in cAMP levels also induces Nocturnin expression, suggesting that Nocturnin's induction in eWAT by fasting is likely mediated through the same pathways that activate lipolysis. Therefore, this suggests that Nocturnin plays a role in linking nutrient sensing by the circadian clock to lipid mobilization in the adipocytes.

Show MeSH
Related in: MedlinePlus