OBJECTIVEResveratrol, a natural polyphenolic compound that is found in grapes and red wine, increases metabolic rate, insulin sensitivity, mitochondrial biogenesis, and physical endurance and reduces fat accumulation in mice. Although it is thought that resveratrol targets Sirt1, this is controversial because resveratrol also activates 5′ AMP-activated protein kinase (AMPK), which also regulates insulin sensitivity and mitochondrial biogenesis. Here, we use mice deficient in AMPKα1 or -α2 to determine whether the metabolic effects of resveratrol are mediated by AMPK.RESEARCH DESIGN AND METHODSMice deficient in the catalytic subunit of AMPK (α1 or α2) and wild-type mice were fed a high-fat diet or high-fat diet supplemented with resveratrol for 13 weeks. Body weight was recorded biweekly and metabolic parameters were measured. We also used mouse embryonic fibroblasts deficient in AMPK to study the role of AMPK in resveratrol-mediated effects in vitro.RESULTSResveratrol increased the metabolic rate and reduced fat mass in wild-type mice but not in AMPKα1−/− mice. In the absence of either AMPKα1 or -α2, resveratrol failed to increase insulin sensitivity, glucose tolerance, mitochondrial biogenesis, and physical endurance. Consistent with this, the expression of genes important for mitochondrial biogenesis was not induced by resveratrol in AMPK-deficient mice. In addition, resveratrol increased the NAD-to-NADH ratio in an AMPK-dependent manner, which may explain how resveratrol may activate Sirt1 indirectly.CONCLUSIONSWe conclude that AMPK, which was thought to be an off-target hit of resveratrol, is the central target for the metabolic effects of resveratrol.

Mitochondrial stress releases mitochondrial DNA (mtDNA) into the cytosol, thereby triggering the type Ι interferon (IFN) response. Mitochondrial outer membrane permeabilization, which is required for mtDNA release, has been extensively studied in apoptotic cells, but little is known about its role in live cells. We found that oxidatively stressed mitochondria release short mtDNA fragments via pores formed by the voltage-dependent anion channel (VDAC) oligomers in the mitochondrial outer membrane. Furthermore, the positively charged residues in the N-terminal domain of VDAC1 interact with mtDNA, promoting VDAC1 oligomerization. The VDAC oligomerization inhibitor VBIT-4 decreases mtDNA release, IFN signaling, neutrophil extracellular traps, and disease severity in a mouse model of systemic lupus erythematosus. Thus, inhibiting VDAC oligomerization is a potential therapeutic approach for diseases associated with mtDNA release.

Metformin is one of the most commonly used first line drugs for type II diabetes. Metformin lowers serum glucose levels by activating 5-AMP-activated kinase (AMPK), which maintains energy homeostasis by directly sensing the AMP/ATP ratio. AMPK plays a central role in food intake and energy metabolism through its activities in central nervous system and peripheral tissues. Since food intake and energy metabolism is synchronized to the light-dark (LD) cycle of the environment, we investigated the possibility that AMPK may affect circadian rhythm. We discovered that the circadian period of Rat-1 fibroblasts treated with metformin was shortened by 1 h. One of the regulators of the period length is casein kinase I⑀ (CKI⑀), which by phosphorylating and inducing the degradation of the circadian clock component, mPer2, shortens the period length. AMPK phosphorylates Ser-389 of CKI⑀, resulting in increased CKI⑀ activity and degradation of mPer2. In peripheral tissues, injection of metformin leads to mPer2 degradation and a phase advance in the circadian expression pattern of clock genes in wild-type mice but not in AMPK ␣2 knock-out mice. We conclude that metformin and AMPK have a previously unrecognized role in regulating the circadian rhythm.Animal behavior, including spontaneous locomotion, sleeping, eating, and drinking, follows a 24-h light-dark (LD) 2 cycle of the environment. The master pacemaker for the rhythmic behavior lies in the suprachiasmatic nucleus (SCN) of the hypothalamus (1). The SCN neurons, cued by the LD cycle, orchestrate the circadian rhythms of peripheral clocks that reside in most cells of the body. The pacemaker, both in the SCN and in peripheral tissues, consists of a self-sustaining near 24-h rhythm in the expression of core clock genes. A central component of this pacemaker is the negative-feedback loop, which results from Per and Cry proteins suppressing their own transcription with a precisely timed lag. A key regulator of the period length is casein kinase I⑀ (CKI⑀) (2). CKI⑀ and its homolog CKI␦ regulate the circadian period by phosphorylating mammalian Per proteins (3-6). The role of CKI⑀ in mammalian circadian rhythm is best illustrated by the semidominant mutation in hamster CKI⑀, tau (7). CKI⑀ tau is a highly specific gain-of-function mutation that increases the CKI⑀ kinase activity on Per proteins. CKI⑀-mediated phosphorylation induces proteasome-mediated degradation of Per proteins, leading to circadian phase advance and shortened period length (8).AMPK, by sensing the rise in AMP level under energy-deprived conditions (9), maintains energy homeostasis by stimulating ATP production and suppressing ATP-consuming processes such as synthesis of macromolecules (10). The catalytic subunit of AMPK has two isoforms, ␣1 (11) and ␣2 (12). Mice with a knockout (KO) of either AMPK ␣1 or AMPK ␣2 are viable (13,14), but mice with a KO of both ␣1 and ␣2 are not viable, 3 indicating that the two isoforms have partially redundant functions. In the hypothalamus, AMPK maintains energy homeostasis at...

The mammalian SWI/SNF-like chromatin-remodeling BAF complex plays several important roles in controlling cell proliferation and differentiation. Interferons (IFNs) are key mediators of cellular antiviral and antiproliferative activities. In this report, we demonstrate that the BAF complex is required for the maximal induction of a subset of IFN target genes by alpha IFN (IFN-␣). The BAF complex is constitutively associated with the IFITM3 promoter in vivo and facilitates the chromatin remodeling of the promoter upon IFN-␣ induction. Furthermore, we show that the ubiquitous transcription activator Sp1 interacts with the BAF complex in vivo and augments the BAF-mediated activation of the IFITM3 promoter. Sp1 binds constitutively to the IFITM3 promoter in the absence of the BAF complex, suggesting that it may recruit and/or stabilize the BAF complex binding to the IFITM3 promoter. Our results bring new mechanistic insights into the antiproliferative effects of the chromatin-remodeling BAF complex.Interferons (IFNs) play several fundamental roles in cellular antiviral and antiproliferative activities (35,40). The signal of alpha/beta IFNs (IFN-␣/␤s) is mainly mediated by IFN-stimulated gene factor 3 (ISGF3), a trimeric complex consisting of STAT1, STAT2, and p48 (14,38,44), which binds to IFNstimulated regulatory elements (ISREs) to activate their target genes (6). Transcriptional activation of the IFN-induced genes is required for the actions of the IFNs (4, 40).The genomic DNA in the nucleus is packaged into nucleosomes that are inhibitory to the access of transcription factors to their target sites. Modification of the nucleosomal template is thought necessary to allow transcriptional activation. This modification can be either covalent bond formation by acetylation, phosphorylation, and methylation at the histone tails and/or noncovalent action by ATP-utilizing remodeling enzymes (1,13,17,19,30,36,41,43,47). Whereas the mechanism by which ISGF3 overcomes the chromatin barrier for binding to its targeting sites is not clear, signal transducer and activator of transcription (STAT) proteins have been found to interact with the highly conserved and ubiquitously expressed cyclic AMP response element-binding proteins, CBP/p300 (3, 15, 34, 49, 52). Both CBP and p300 have histone acetyltransferase activity (33). Overexpression of E1A, which binds to CBP/p300 and inhibits its histone acetyltransferase activity, blocks the ISRE-mediated responses to IFN-␣/␤ (3), suggesting that the modification of the chromatin structure might be an important step in the induction of IFN target genes.It was reported recently that the reconstitution of the active BAF complex by transient expression of the essential ATPase subunit BRG1 in BRG1-deficient SW-13 cells up-regulates several IFN target genes (27), suggesting that the chromatinremodeling activity of the BAF complex might be required for the induction of these genes by IFNs. In this report, we demonstrate that reconstitution of the BAF complex by transiently expressing BRG1 in SW-1...

Protein phosphatase 5 (PP5) exhibits low basal activity due to the autoinhibitory properties of its N-terminal and C-terminal domains but can be activated approximately 40-fold in vitro by polyunsaturated fatty acids. To identify residues involved in regulating PP5 activity, we performed scanning mutagenesis of its N-terminal tetratricopeptide repeat (TPR) domain and deletion mutagenesis of its C-terminal domain. Mutating residues in a groove of the TPR domain that binds to heat shock protein 90 had no effect on basal phosphatase activity. Mutation of Glu-76, however, whose side chain projects away from this groove, resulted in a 10-fold elevation of basal activity without affecting arachidonic acid-stimulated activity. Thus, the interface of the TPR domain involved in PP5 autoinhibition appears to be different from that involved in heat shock protein 90 binding. We also observed a 10-fold elevation of basal phosphatase activity upon removing the C-terminal 13 amino acids of PP5, with a concomitant 50% decrease in arachidonic acid-stimulated activity. These two effects were accounted for by two distinct amino acid deletions: deleting the four C-terminal residues (496-499) of PP5 had no effect on its activity, but removing Gln-495 elevated basal activity 10-fold. Removal of a further three amino acids had no additional effect, but deleting Asn-491 resulted in a 50% reduction in arachidonic acid-stimulated activity. Thus, Glu-76 in the TPR domain and Gln-495 at the C-terminus were implicated in maintaining the low basal activity of PP5. While the TPR domain alone has been thought to mediate fatty acid activation of PP5, our data suggest that Asn-491, near its C-terminus, may also be involved in this process.

SIRT1, an NAD+ (nicotinamide adenine dinucleotide)-dependent deacetylase, protects cells from stress-induced apoptosis, and its orthologues delay aging in lower eukaryotes. SIRT1 increases survival in response to stress such as DNA damage by deacetylating a number of substrates including pro-apoptotic protein p53. The molecular mechanism by which DNA-damage activates SIRT1 is not known. By screening a kinase inhibitor library, we identified CK2 as a SIRT1 kinase. CK2 is a pleiotropic kinase with more than 300 substrates and well-known anti-apoptotic and pro-growth activities. We find that CK2 is recruited to SIRT1 after ionizing radiation (IR) and phosphorylates conserved residues Ser 154, 649, 651 and 683 in the N- and C-terminal domains of mouse SIRT1. Phosphorylation of SIRT1 increases its deacetylation rate but not if the four Ser residues are mutated. In addition, phosphorylation of SIRT1 increases its substrate-binding affinity. CK2-mediated phosphorylation increases the ability of SIRT1 to deacetylate p53 and protect cells from apoptosis after DNA damage. Based on these findings, we propose that CK2 protects against IR-induced apoptosis partly by phosphorylating and activating SIRT1. Thus, this work suggests that SIRT1 is a component of the expansive anti-apoptotic network controlled by CK2. Since expression of both CK2 and SIRT1 is upregulated with tumorigenesis and downregulated with senescence, the CK2-SIRT1 link sheds new light on how CK2 may regulate cancer development and aging.

The ubiquitous mammalian chromatin-remodeling SWI/SNF-like BAF complexes play critical roles in tumorigenesis. It was suggested that the direct interaction of BRG1 with the retinoblastoma protein pRB is required for regulation of cell cycle progression by pRB. We present evidence that the BRG1-containing complexes regulate the expression of the cdk inhibitor p21 CIP1/WAF1/SDI . Furthermore, we show that the physical interaction between BRG1 and pRB is not required for induction of cell growth arrest and transcriptional repression of E2F target genes by pRB. Instead, BRG1 activates pRB by inducing its hypophosphorylation through up-regulation of the cdk inhibitor p21. The hypophosphorylation of pRB is reinforced by down-regulation of critical components, including cdk2, cyclin E, and cyclin D, in the pRB regulatory network. We demonstrate that up-regulation of p21 by BRG1 is necessary to induce formation of flat cells, growth arrest, and finally, cell senescence. Our results suggest that the BRG1-containing complexes control cellular proliferation and senescence by modulating the pRB pathway via multiple mechanisms.Increasing genetic evidence indicates that the mammalian chromatin-remodeling SWI/SNF-like BAF or hSWI/SNF complexes (28,32,66) play an important role in controlling cell proliferation and differentiation and in inhibiting cancer formation (reviewed in reference 35). Various homozygous mutations in the INI1/hSNF5/BAF47 subunit are linked to malignant rhabdoid tumors (MRTs), which are aggressive pediatric tumors in children under 5 years of age (14,53,65). Mouse models with targeted disruptions of the BAF complex have also provided evidence supporting a role for the BAF complexes in inhibiting tumorigenesis. While homozygous deletions of the INI1/BAF47 gene in mouse are embryonic lethal, the heterozygous mice develop tumors at a high frequency (23,34,49). Similarly, homozygous deletions of BRG1, the essential ATPase subunit of the BAF complex, are lethal, while the heterozygous mice are predisposed to cancer formation (8). Interestingly, reintroduction of BRG1 into SW-13 cells that do not express detectable levels of BRG1 is sufficient to reverse their transformed phenotype by inducing growth arrest and a flattened shape, which requires the activity of the retinoblastoma protein (pRB) (17).pRB is a major tumor suppressor that is frequently disrupted in cancer cells (55). It is a nuclear phosphoprotein that arrests cells in G 0 /G 1 phase by repressing genes required for the G 1 /S phase transition (68). The transcriptional repression by pRB is mediated by interaction with the E2F family of transcription factors, whose binding sites are found in the promoters of many genes involved in cell cycle progression (reviewed in references 18 and 47). The interaction of pRB with E2F is controlled by the phosphorylation status of several serine and threonine residues. Inactivation of pRB by phosphorylation releases E2F and therefore the repression of its target genes, which allows the cell cycle to progress throug...

In mammals, the Sirtuins are composed of seven Sir2 orthologues (Sirt1-7) with a conserved deacetylase core that utilizes NAD+ as a cofactor. Interestingly, the deacetylase core of Sirt1 by itself has no catalytic activity. We found within the C-terminal domain a 25 a.a. sequence that is essential for Sirt1 activity (ESA). Our results indicate that the ESA region interacts with and functions as an “on switch” for the deacetylase core. The endogenous Sirt1 inhibitor DBC1, which also binds to the deacetylase core, competes with and inhibits the ESA region from interacting with the deacetylase core. We discovered an ESA mutant peptide that can bind to the deacetylase core and inhibit Sirt1 in trans. By using this mutant peptide, we were able to inhibit Sirt1 activity and to increase the chemosensitivity of androgen-refractory prostate cancer cells. Therefore, the ESA region is a potential target for development of therapies to regulate Sirt1.