Ultradian rhythms of AKT phosphorylation and gene expression emerge in the absence of the circadian clock components Per1 and Per2

Rhythmicity of biological processes can be elicited either in response to environmental cycles or driven by endogenous oscillators. In mammals, the circadian clock drives about 24-hour rhythms of multitude metabolic and physiological processes in anticipation to environmental daily oscillations. Also at the intersection of environment and metabolism is the protein kinase—AKT. It conveys extracellular signals, primarily feeding-related signals, to regulate various key cellular functions. Previous studies in mice identified rhythmicity in AKT activation (pAKT) with elevated levels in the fed state. However, it is still unknown whether rhythmic AKT activation can be driven through intrinsic mechanisms. Here, we inspected temporal changes in pAKT levels both in cultured cells and animal models. In cultured cells, pAKT levels showed circadian oscillations similar to those observed in livers of wild-type mice under free-running conditions. Unexpectedly, in livers of Per1,2−/− but not of Bmal1−/− mice we detected ultradian (about 16 hours) oscillations of pAKT levels. Importantly, the liver transcriptome of Per1,2−/− mice also showed ultradian rhythms, corresponding to pAKT rhythmicity and consisting of AKT-related genes and regulators. Overall, our findings reveal ultradian rhythms in liver gene expression and AKT phosphorylation that emerge in the absence of environmental rhythms and Per1,2−/− genes.

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Lanthionine synthetase C–like protein 2 (LanCL2) is a novel regulator of Akt

Molecular Biology of the Cell ◽

10.1091/mbc.e14-01-0004 ◽

2014 ◽

Vol 25 (24) ◽

pp. 3954-3961 ◽

Cited By ~ 30

Author(s):

Min Zeng ◽

Wilfred A. van der Donk ◽

Jie Chen

Keyword(s):

Insulin Receptor ◽

Insulin Receptor Substrate ◽

Akt Phosphorylation ◽

Akt Activation ◽

Cellular Processes ◽

Human Liver Cells ◽

Wide Range ◽

Physical Interactions ◽

Protein Kinase Akt

The serine/threonine protein kinase Akt controls a wide range of biochemical and cellular processes under the modulation of a variety of regulators. In this study, we identify the lanthionine synthetase C–like 2 (LanCL2) protein as a positive regulator of Akt activation in human liver cells. LanCL2 knockdown dampens serum- and insulin-stimulated Akt phosphorylation, whereas LanCL2 overexpression enhances these processes. Neither insulin receptor phosphorylation nor the interaction between insulin receptor substrate and phosphatidylinositide 3-kinase (PI3K) is affected by LanCL2 knockdown. LanCL2 also does not function through PP2A, a phosphatase of Akt. Instead, LanCL2 directly interacts with Akt, with a preference for inactive Akt. Moreover, we show that LanCL2 also binds to the Akt kinase mTORC2, but not phosphoinositide-dependent kinase 1. Whereas LanCL2 is not required for the Akt-mTORC2 interaction, recombinant LanCL2 enhances Akt phosphorylation by target of rapamycin complex 2 (mTORC2) in vitro. Finally, consistent with a function of Akt in regulating cell survival, LanCL2 knockdown increases the rate of apoptosis, which is reversed by the expression of a constitutively active Akt. Taken together, our findings reveal LanCL2 as a novel regulator of Akt and suggest that LanCL2 facilitates optimal phosphorylation of Akt by mTORC2 via direct physical interactions with both the kinase and the substrate.

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The Circadian NAD+Metabolism: Impact on Chromatin Remodeling and Aging

BioMed Research International ◽

10.1155/2016/3208429 ◽

2016 ◽

Vol 2016 ◽

pp. 1-7 ◽

Cited By ~ 11

Author(s):

Yasukazu Nakahata ◽

Yasumasa Bessho

Keyword(s):

Gene Expression ◽

Circadian Clock ◽

Cell Fate ◽

Chromatin Remodeling ◽

Expression Patterns ◽

Fine Tuning ◽

Cellular Functions ◽

Nad Metabolism ◽

Structure Changes ◽

Age Related

Gene expression is known to be a stochastic phenomenon. The stochastic gene expression rate is thought to be altered by topological change of chromosome and/or by chromatin modifications such as acetylation and methylation. Changes in mechanical properties of chromosome/chromatin by soluble factors, mechanical stresses from the environment, or metabolites determine cell fate, regulate cellular functions, or maintain cellular homeostasis. Circadian clock, which drives the expression of thousands of genes with 24-hour rhythmicity, has been known to be indispensable for maintaining cellular functions/homeostasis. During the last decade, it has been demonstrated that chromatin also undergoes modifications with 24-hour rhythmicity and facilitates the fine-tuning of circadian gene expression patterns. In this review, we cover data which suggests that chromatin structure changes in a circadian manner and that NAD+is the key metabolite for circadian chromatin remodeling. Furthermore, we discuss the relationship among circadian clock, NAD+metabolism, and aging/age-related diseases. In addition, the interventions of NAD+metabolism for the prevention and treatment of aging and age-related diseases are also discussed.

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Estradiol-induced regulation of GLUT4 in 3T3-L1 cells: involvement of ESR1 and AKT activation

Journal of Molecular Endocrinology ◽

10.1530/jme-17-0041 ◽

2017 ◽

Vol 59 (3) ◽

pp. 257-268 ◽

Cited By ~ 5

Author(s):

Raquel S Campello ◽

Luciana A Fátima ◽

João Nilton Barreto-Andrade ◽

Thais F Lucas ◽

Rosana C Mori ◽

...

Keyword(s):

Plasma Membrane ◽

Glucose Uptake ◽

Subcellular Distribution ◽

Glucose Transporter ◽

Biological Effects ◽

Glut4 Translocation ◽

Akt Phosphorylation ◽

Akt Activation ◽

Glut4 Expression ◽

Protein Kinase Akt

Impaired insulin-stimulated glucose uptake involves reduced expression of the GLUT4 (solute carrier family 2 facilitated glucose transporter member 4, SLC2A4 gene). 17β-estradiol (E2) modulates SLC2A4/GLUT4 expression, but the involved mechanisms are unclear. Although E2 exerts biological effects by binding to estrogen receptors 1/2 (ESR1/2), which are nuclear transcriptional factors; extranuclear effects have also been proposed. We hypothesize that E2 regulates GLUT4 through an extranuclear ESR1 mechanism. Thus, we investigated the effects of E2 upon (1) subcellular distribution of ESRs and the proto-oncogene tyrosine-protein kinases (SRC) involvement; (2) serine/threonine-protein kinase (AKT) activation; (3) Slc2a4/GLUT4 expression and (4) GLUT4 subcellular distribution and glucose uptake in 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were cultivated or not with E2 for 24 h, and additionally treated or not with ESR1-selective agonist (PPT), ESR1-selective antagonist (MPP) or selective SRC inhibitor (PP2). Subcellular distribution of ESR1, ESR2 and GLUT4 was analyzed by immunocytochemistry; Slc2a4 mRNA and GLUT4 were quantified by qPCR and Western blotting, respectively; plasma membrane GLUT4 translocation and glucose uptake were analyzed under insulin stimulus for 20 min or not. E2 induced (1) translocation of ESR1, but not of ESR2, from nucleus to plasma membrane and AKT phosphorylation, effects mimicked by PPT and blocked by MPP and PP2; (2) increased Slc2a4/GLUT4 expression and (3) increased insulin-stimulated GLUT4 translocation and glucose uptake. In conclusion, E2 treatment promoted a SRC-mediated nucleus-plasma membrane shuttle of ESR1, and increased AKT phosphorylation, Slc2a4/GLUT4 expression and plasma membrane GLUT4 translocation; consequently, improving insulin-stimulated glucose uptake. These results unravel mechanisms through which estrogen improves insulin sensitivity.

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Structural basis for transcriptional coactivator recognition by SMAD2 in TGF-β signaling

Science Signaling ◽

10.1126/scisignal.abb9043 ◽

2020 ◽

Vol 13 (662) ◽

pp. eabb9043

Author(s):

Ken-ichi Miyazono ◽

Tomoko Ito ◽

Yui Fukatsu ◽

Hikaru Wada ◽

Akira Kurisaki ◽

...

Keyword(s):

Gene Expression ◽

Transforming Growth Factor ◽

Cultured Cells ◽

Hydrophobic Surface ◽

Transforming Growth Factor Β ◽

Structural Basis ◽

Transcriptional Coactivator ◽

Cellular Functions ◽

Biochemical Basis ◽

Smad Proteins

Transforming growth factor–β (TGF-β) proteins regulate multiple cellular functions, including cell proliferation, apoptosis, and extracellular matrix formation. The dysregulation of TGF-β signaling causes diseases such as cancer and fibrosis, and therefore, understanding the biochemical basis of TGF-β signal transduction is important for elucidating pathogenic mechanisms in these diseases. SMAD proteins are transcription factors that mediate TGF-β signaling–dependent gene expression. The transcriptional coactivator CBP directly interacts with the MH2 domains of SMAD2 to activate SMAD complex–dependent gene expression. Here, we report the structural basis for CBP recognition by SMAD2. The crystal structures of the SMAD2 MH2 domain in complex with the SMAD2-binding region of CBP showed that CBP forms an amphiphilic helix on the hydrophobic surface of SMAD2. The expression of a mutated CBP peptide that showed increased SMAD2 binding repressed SMAD2-dependent gene expression in response to TGF-β signaling in cultured cells. Disrupting the interaction between SMAD2 and CBP may therefore be a promising strategy for suppressing SMAD-dependent gene expression.

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Greater insulin-mediated Akt phosphorylation concomitant with heterogeneous effects on phosphorylation of Akt substrates in soleus of calorie-restricted rats

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00457.2012 ◽

2012 ◽

Vol 303 (12) ◽

pp. R1261-R1267 ◽

Cited By ~ 10

Author(s):

Naveen Sharma ◽

Donel A. Sequea ◽

Edward B. Arias ◽

Gregory D. Cartee

Keyword(s):

Protein Synthesis ◽

Insulin Signaling ◽

Cellular Growth ◽

Alternative Mechanism ◽

Cellular Functions ◽

Akt Phosphorylation ◽

Akt Activation ◽

Heterogeneous Effects ◽

Signaling Events ◽

Old Rats

Akt is a serine/threonine kinase that plays a key role in numerous cellular functions including metabolism, growth, protein synthesis, apoptosis, and cell proliferation. The most consistent and robust effect of moderate calorie restriction (CR; ∼60% of ad libitum, AL, food consumption) on insulin signaling in rodent muscle has been enhanced insulin-induced phosphorylation of Akt (pAkt). However, there is limited knowledge regarding the mechanism for this enhancement and its consequences in predominantly slow-twitch muscle. Accordingly, in soleus muscle of 9-mo-old rats, we analyzed the effect of CR and insulin on important signaling events that are proximal to Akt activation including: pIRTyr1162/1163, pIRS1Tyr, pIRS1Ser312, IRS1-associated phosphatidylinositol 3-kinase activity, or pPTENSer380. In addition, we analyzed the effect of CR and insulin on Akt substrates that have established or putative roles in glucose metabolism, cellular growth, maintenance of muscle structure, or protein synthesis including pGSK3αSer21, pGSK3βSer9, pTSC2Ser939, pP70S6KThr412, pAS160Thr642, and pFLNcSer2213. The current study demonstrated that the CR-induced increase in pAkt in isolated soleus muscles from 9-mo-old rats can occur without concomitant enhancement of several important insulin signaling events that are proximal to Akt activation. These results suggest that the greater pAkt in the soleus muscles from CR rats was attributable to an alternative mechanism. We also observed that the effects of CR were not uniform for phosphorylation of six insulin-regulated Akt substrates in the soleus. The differential response in phosphorylation by Akt substrates likely has important implications for explaining the complex effect of CR diverse cellular functions.

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“A novel female-specific circadian clock mechanism regulating metabolism”

10.1101/2020.10.05.326652 ◽

2020 ◽

Author(s):

Tsedey Mekbib ◽

Ting-Chung Suen ◽

Aisha Rollins-Hairston ◽

Kiandra Smith ◽

Ariel Armstrong ◽

...

Keyword(s):

Gene Expression ◽

Circadian Clock ◽

Cultured Cells ◽

Clock Genes ◽

Cholesterol Metabolism ◽

Lipoprotein Metabolism ◽

Circadian Clocks ◽

Control Of Gene Expression ◽

Female Specific

AbstractCircadian clocks enable organisms to predict and align their behaviors and physiologies to constant daily day-night environmental cycle. Because the ubiquitin ligase Siah2 has been identified as a potential regulator of circadian clock function in cultured cells, we have used Siah2-deficient mice to examine its function in vivo. Our experiments demonstrate a striking and unexpected sexually dimorphic effect of Siah2 deficiency on the regulation of rhythmically expressed genes. The absence of Siah2 in females, but not in males, altered the expression of core circadian clock genes and drastically remodeled the rhythmic hepatic transcriptome. Siah2 loss, only in females, increased the expression of 100’s of genes selectively at mid-day, resulting in a ∼70% increase in the number of rhythmically expressed genes, and shifted the expression of 100’s of other genes from a mid-night peak, to a mid-day peak. The combined result is a near inversion of overall rhythmicity in gene expression selectively in Siah2-deficient females. This dramatic reorganization created a substantial misalignment between rhythmic liver functions and feeding/behavioral rhythms, and consequently impaired daily patterns of lipid/lipoprotein metabolism and metabolic responses to high-fat diet. Collectively, our results suggest that Siah2 is part of a female-specific circadian mechanism important for maintaining metabolic homeostasis and may play a key role in the establishing sexual dimorphisms in metabolism.Signficance statementCircadian clocks drive daily rhythms in many aspects of our physiology, optimally aligning functions across the day-night cycle. How circadian clocks drives these rhythms is thought to be due to largely similar transcriptional pathways and mechanisms in males and females, although some rhythms are modulated by sex and growth hormones. In this study, we present data that uncover the surprising existence of a female-specific transcriptional mechanism that is essential for the proper rhythmic control of gene expression in the liver. Disrupting this mechanism substantially impairs the circadian regulation of lipid and cholesterol metabolism selectively in females, impairing their resistance to diet-induced obesity. These results reveal that circadian clocks may be broadly coupled to physiological rhythms using unexpected sex-specific mechanisms.

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Regulation of the poly(ADP-ribose) polymerase-1 gene expression by the transcription factors Sp1 and Sp3 is under the influence of cell density in primary cultured cells

Biochemical Journal ◽

10.1042/bj20041718 ◽

2005 ◽

Vol 389 (2) ◽

pp. 423-433 ◽

Cited By ~ 15

Author(s):

Karine Zaniolo ◽

Anne Rufiange ◽

Steeve Leclerc ◽

Serge Desnoyers ◽

Sylvain L. Guérin

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Western Blot ◽

Cell Density ◽

Cultured Cells ◽

Gene Promoter ◽

Cellular Functions ◽

Mobility Shift ◽

Cell Densities ◽

Parp 1

PARP-1 [poly(ADP-ribose) polymerase-1) is a nuclear enzyme that is involved in several cellular functions, including DNA repair, DNA transcription, carcinogenesis and apoptosis. The activity directed by the PARP-1 gene promoter is mainly dictated through its recognition by the transcription factors Sp1 and Sp3 (where Sp is specificity protein). In the present study, we investigated whether (i) both PARP-1 expression and PARP-1 enzymatic activity are under the influence of cell density in primary cultured cells, and (ii) whether its pattern of expression is co-ordinated with that of Sp1/Sp3 at varying cell densities and upon cell passages. All types of cultured cells expressed PARP-1 in Western blot when grown to sub-confluence. However, a dramatic reduction was observed at post-confluence. Similarly, high levels of Sp1/Sp3 were observed by both Western blot and EMSAs (electrophoretic mobility-shift assays) in sub-confluent, but not post-confluent, cells. Consistent with these results, the promoter of the rPARP-1 (rat PARP-1) gene directed high levels of activity in sub-confluent, but not confluent, cells upon transfection of various CAT (chloramphenicol acetyltransferase)–rPARP-1 promoter constructs into cultured cells. The positive regulatory influence of Sp1 was not solely exerted on the rPARP-1 promoter constructs, as inhibition of endogenous Sp1 expression in HDKs (human dermal keratinocytes) through the transfection of Sp1 RNAi (RNA interference) considerably reduced endogenous hPARP-1 (human PARP-1) expression as well. The reduction in PARP-1 protein expression as cells reached confluence also translated into a corresponding reduction in PARP-1 activity. In addition, expression of both Sp1/Sp3, as well as that of PARP-1, was dramatically reduced as cells were passaged in culture and progressed towards irreversible terminal differentiation. PARP-1 gene expression therefore appears to be co-ordinated with that of Sp1 and Sp3 in primary cultured cells, suggesting that PARP-1 may play some important functions during the proliferative burst that characterizes wound healing.

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