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Knock-in Luciferase Reporter Mice for In Vivo Monitoring of CREB Activity.

Akhmedov D, Rajendran K, Mendoza-Rodriguez MG, Berdeaux R - PLoS ONE (2016)

Bottom Line: To overcome disadvantages of the currently used strategies, we created a ROSA26 knock-in CREB reporter mouse line (ROSA26-CRE-luc). cAMP-inducing ligands stimulate the reporter in primary hepatocytes and myocytes from ROSA26-CRE-luc animals.In vivo, these animals exhibit little hepatic CREB activity in the ad libitum fed state but robust induction after fasting.The ROSA26-CRE-luc mouse line is a useful resource to study dynamics of CREB activity longitudinally in vivo and can be used as a source of primary cells for analysis of CREB regulatory pathways ex vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Biology and Pharmacology, McGovern Medical School at the University of Texas Health Science Center at Houston (UTHealth), Houston, Texas, United States of America.

ABSTRACT
The cAMP response element binding protein (CREB) is induced during fasting in the liver, where it stimulates transcription of rate-limiting gluconeogenic genes to maintain metabolic homeostasis. Adenoviral and transgenic CREB reporters have been used to monitor hepatic CREB activity non-invasively using bioluminescence reporter imaging. However, adenoviral vectors and randomly inserted transgenes have several limitations. To overcome disadvantages of the currently used strategies, we created a ROSA26 knock-in CREB reporter mouse line (ROSA26-CRE-luc). cAMP-inducing ligands stimulate the reporter in primary hepatocytes and myocytes from ROSA26-CRE-luc animals. In vivo, these animals exhibit little hepatic CREB activity in the ad libitum fed state but robust induction after fasting. Strikingly, CREB was markedly stimulated in liver, but not in skeletal muscle, after overnight voluntary wheel-running exercise, uncovering differential regulation of CREB in these tissues under catabolic states. The ROSA26-CRE-luc mouse line is a useful resource to study dynamics of CREB activity longitudinally in vivo and can be used as a source of primary cells for analysis of CREB regulatory pathways ex vivo.

No MeSH data available.


Related in: MedlinePlus

Hepatic CREB is activated after voluntary exercise.(A) Bioluminescence imaging of female ROSA26-CRE-luc knock-in mice (n = 6), static housed (0 h run, ZT10) and after 12 h voluntary wheel running exercise (ZT12-day2 ZT0) (5 sec exposure). (B) Quantification of hepatic bioluminescence in ROSA26-CRE-luc mice shown in A (n = 6, median, 25th and 75th percentile and range indicated). (C) Luciferase activity, (D) Western blot of phospho-CREB(S133)/ATF-1 and total CREB, (E) Quantification of pCREB western blots and (F) G6Pase and PGC-1α mRNA normalized to Gapdh from liver of mice run for 0 and 12 h (n = 6, mean ±SEM). *p<0.05, **p<0.01, ***p<0.001 by t-tests.
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pone.0158274.g002: Hepatic CREB is activated after voluntary exercise.(A) Bioluminescence imaging of female ROSA26-CRE-luc knock-in mice (n = 6), static housed (0 h run, ZT10) and after 12 h voluntary wheel running exercise (ZT12-day2 ZT0) (5 sec exposure). (B) Quantification of hepatic bioluminescence in ROSA26-CRE-luc mice shown in A (n = 6, median, 25th and 75th percentile and range indicated). (C) Luciferase activity, (D) Western blot of phospho-CREB(S133)/ATF-1 and total CREB, (E) Quantification of pCREB western blots and (F) G6Pase and PGC-1α mRNA normalized to Gapdh from liver of mice run for 0 and 12 h (n = 6, mean ±SEM). *p<0.05, **p<0.01, ***p<0.001 by t-tests.

Mentions: During exercise, glucose is rapidly utilized by skeletal muscle, creating a catabolic state. To compensate for glucose utilization and maintain glucose homeostasis during exercise, glucagon and catecholamines are released and, in turn, stimulate hepatic glucose production by glycogenolysis and gluconeogenesis [22, 23]. Liver expression of gluconeogenic genes encoding phosphoenol pyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) increases within hours of running initiation in rodents [24–26]. As CREB is known to directly regulate transcription of G6Pase as well as the transcriptional regulator Pgc-1α in liver [2, 27, 28] and CREB phosphorylation in muscle has been shown to increase after 30 minutes of strenuous exercise [29], we tested CREB activity in female ROSA26-CRE-luc mice at baseline (static housing) and after 12 h of voluntary running during the dark cycle. We observed approximately 30-fold increased CREB reporter bioluminescence in liver of mice after 12 h voluntary running compared to the same mice measured before exercise (Fig 2A and 2B) and 10-fold increased bioluminescence emanating from the brain (Figs 2A and S2A). Accordingly, biochemical luciferase activity was increased in liver lysates of exercised mice compared with static housed controls (Figs 2C and S2B) and was correlated with hepatic in vivo bioluminescence signals (S2C Fig). CREB phosphorylation on Ser133, which is required for CREB activity [30], was increased about 2.5-fold in liver after 12 h of exercise (Fig 2D and 2E). Supporting these results, G6Pase and PGC-1α mRNAs were increased in liver of exercised mice relative to static housed controls (Fig 2F).


Knock-in Luciferase Reporter Mice for In Vivo Monitoring of CREB Activity.

Akhmedov D, Rajendran K, Mendoza-Rodriguez MG, Berdeaux R - PLoS ONE (2016)

Hepatic CREB is activated after voluntary exercise.(A) Bioluminescence imaging of female ROSA26-CRE-luc knock-in mice (n = 6), static housed (0 h run, ZT10) and after 12 h voluntary wheel running exercise (ZT12-day2 ZT0) (5 sec exposure). (B) Quantification of hepatic bioluminescence in ROSA26-CRE-luc mice shown in A (n = 6, median, 25th and 75th percentile and range indicated). (C) Luciferase activity, (D) Western blot of phospho-CREB(S133)/ATF-1 and total CREB, (E) Quantification of pCREB western blots and (F) G6Pase and PGC-1α mRNA normalized to Gapdh from liver of mice run for 0 and 12 h (n = 6, mean ±SEM). *p<0.05, **p<0.01, ***p<0.001 by t-tests.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4940169&req=5

pone.0158274.g002: Hepatic CREB is activated after voluntary exercise.(A) Bioluminescence imaging of female ROSA26-CRE-luc knock-in mice (n = 6), static housed (0 h run, ZT10) and after 12 h voluntary wheel running exercise (ZT12-day2 ZT0) (5 sec exposure). (B) Quantification of hepatic bioluminescence in ROSA26-CRE-luc mice shown in A (n = 6, median, 25th and 75th percentile and range indicated). (C) Luciferase activity, (D) Western blot of phospho-CREB(S133)/ATF-1 and total CREB, (E) Quantification of pCREB western blots and (F) G6Pase and PGC-1α mRNA normalized to Gapdh from liver of mice run for 0 and 12 h (n = 6, mean ±SEM). *p<0.05, **p<0.01, ***p<0.001 by t-tests.
Mentions: During exercise, glucose is rapidly utilized by skeletal muscle, creating a catabolic state. To compensate for glucose utilization and maintain glucose homeostasis during exercise, glucagon and catecholamines are released and, in turn, stimulate hepatic glucose production by glycogenolysis and gluconeogenesis [22, 23]. Liver expression of gluconeogenic genes encoding phosphoenol pyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) increases within hours of running initiation in rodents [24–26]. As CREB is known to directly regulate transcription of G6Pase as well as the transcriptional regulator Pgc-1α in liver [2, 27, 28] and CREB phosphorylation in muscle has been shown to increase after 30 minutes of strenuous exercise [29], we tested CREB activity in female ROSA26-CRE-luc mice at baseline (static housing) and after 12 h of voluntary running during the dark cycle. We observed approximately 30-fold increased CREB reporter bioluminescence in liver of mice after 12 h voluntary running compared to the same mice measured before exercise (Fig 2A and 2B) and 10-fold increased bioluminescence emanating from the brain (Figs 2A and S2A). Accordingly, biochemical luciferase activity was increased in liver lysates of exercised mice compared with static housed controls (Figs 2C and S2B) and was correlated with hepatic in vivo bioluminescence signals (S2C Fig). CREB phosphorylation on Ser133, which is required for CREB activity [30], was increased about 2.5-fold in liver after 12 h of exercise (Fig 2D and 2E). Supporting these results, G6Pase and PGC-1α mRNAs were increased in liver of exercised mice relative to static housed controls (Fig 2F).

Bottom Line: To overcome disadvantages of the currently used strategies, we created a ROSA26 knock-in CREB reporter mouse line (ROSA26-CRE-luc). cAMP-inducing ligands stimulate the reporter in primary hepatocytes and myocytes from ROSA26-CRE-luc animals.In vivo, these animals exhibit little hepatic CREB activity in the ad libitum fed state but robust induction after fasting.The ROSA26-CRE-luc mouse line is a useful resource to study dynamics of CREB activity longitudinally in vivo and can be used as a source of primary cells for analysis of CREB regulatory pathways ex vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Biology and Pharmacology, McGovern Medical School at the University of Texas Health Science Center at Houston (UTHealth), Houston, Texas, United States of America.

ABSTRACT
The cAMP response element binding protein (CREB) is induced during fasting in the liver, where it stimulates transcription of rate-limiting gluconeogenic genes to maintain metabolic homeostasis. Adenoviral and transgenic CREB reporters have been used to monitor hepatic CREB activity non-invasively using bioluminescence reporter imaging. However, adenoviral vectors and randomly inserted transgenes have several limitations. To overcome disadvantages of the currently used strategies, we created a ROSA26 knock-in CREB reporter mouse line (ROSA26-CRE-luc). cAMP-inducing ligands stimulate the reporter in primary hepatocytes and myocytes from ROSA26-CRE-luc animals. In vivo, these animals exhibit little hepatic CREB activity in the ad libitum fed state but robust induction after fasting. Strikingly, CREB was markedly stimulated in liver, but not in skeletal muscle, after overnight voluntary wheel-running exercise, uncovering differential regulation of CREB in these tissues under catabolic states. The ROSA26-CRE-luc mouse line is a useful resource to study dynamics of CREB activity longitudinally in vivo and can be used as a source of primary cells for analysis of CREB regulatory pathways ex vivo.

No MeSH data available.


Related in: MedlinePlus