Myung K. Kim

Resveratrol ameliorates aging-related metabolic phenotypes by inhibiting cAMP phosphodiesterases

Resveratrol, a polyphenol in red wine, has been reported as a calorie restriction mimetic with potential antiaging and antidiabetogenic properties. It is widely consumed as a nutritional supplement, but its mechanism of action remains a mystery. Here, we report that the metabolic effects of resveratrol result from competitive inhibition of cAMP-degrading phosphodiesterases, leading to elevated cAMP levels. The resulting activation of Epac1, a cAMP effector protein, increases intracellular Ca(2+) levels and activates the CamKKβ-AMPK pathway via phospholipase C and the ryanodine receptor Ca(2+)-release channel. As a consequence, resveratrol increases NAD(+) and the activity of Sirt1. Inhibiting PDE4 with rolipram reproduces all of the metabolic benefits of resveratrol, including prevention of diet-induced obesity and an increase in mitochondrial function, physical stamina, and glucose tolerance in mice. Therefore, administration of PDE4 inhibitors may also protect against and ameliorate the symptoms of metabolic diseases associated with aging.

AMP-Activated Protein Kinase–Deficient Mice Are Resistant to the Metabolic Effects of Resveratrol

OBJECTIVE Resveratrol, 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 METHODS Mice 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. RESULTS Resveratrol 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. CONCLUSIONS We conclude that AMPK, which was thought to be an off-target hit of resveratrol, is the central target for the metabolic effects of resveratrol.

PML-dependent apoptosis after DNA damage is regulated by the checkpoint kinase hCds1/Chk2.

The promyelocytic leukaemia (PML) gene is translocated in most acute promyelocytic leukaemias and encodes a tumour suppressor protein. PML is involved in multiple apoptotic pathways and is thought to be pivotal in γ irradiation-induced apoptosis. The DNA damage checkpoint kinase hCds1/Chk2 is necessary for p53-dependent apoptosis after γ irradiation. In addition, γ irradiation-induced apoptosis also occurs through p53-independent mechanisms, although the molecular mechanism remains largely unknown. Here, we report that hCds1/Chk2 mediates γ irradiation-induced apoptosis in a p53-independent manner through an ataxia telangiectasia-mutated (ATM)–hCds1/Chk2–PML pathway. Our results provide the first evidence of a functional relationship between PML and a checkpoint kinase in γ irradiation-induced apoptosis.

The Role of mPer2 Clock Gene in Glucocorticoid and Feeding Rhythms

The circadian clock synchronizes the activity level of an organism to the light-dark cycle of the environment. Energy intake, as well as energy metabolism, also has a diurnal rhythm. Although the role of the clock genes in the sleep-wake cycle is well characterized, their role in the generation of the metabolic rhythms is poorly understood. Here, we use mice deficient in the clock protein mPer2 to study how the circadian clock regulates two critical metabolic rhythms: glucocorticoid and food intake rhythms. Our findings indicate that mPer2-/- mice do not have a glucocorticoid rhythm even though the corticosterone response to hypoglycemia, ACTH, and restraint stress is intact. In addition, the diurnal feeding rhythm is absent in mPer2-/- mice. On high-fat diet, they eat as much during the light period as they do during the dark period and develop significant obesity. The diurnal rhythm of neuroendocrine peptide alphaMSH, a major effector of appetite control, is disrupted in the hypothalamus of mPer2-/- mice even though the diurnal rhythm of ACTH, the alphaMSH precursor, is intact. Peripheral injection of alphaMSH, which has been shown to enter the brain, restored the feeding rhythm and induced weight loss in mPer2-/- mice. These findings emphasize the requirement of mPer2 in appetite control during the inactive period and the potential role of peripherally administered alphaMSH in restoring night-day eating pattern in individuals with circadian eating disorders such as night-eating syndrome, which is also associated with obesity.

CK2 is the regulator of SIRT1 substrate-binding affinity, deacetylase activity and cellular response to DNA-damage.

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.

A soluble transcription factor, Oct-1, is also found in the insoluble nuclear matrix and possesses silencing activity in its alanine-rich domain.

Expression of the human thyrotropin beta (hTSHbeta) gene is restricted to thyrotrophs, at least in part, by silencing. Using transient-transfection assays, we have localized a silencer element to a region between -128 and -480 bp upstream of the transcription initiation site. The silencing activity was overcome in a thyrotroph-specific manner by an unknown enhancer located in the sequences at -approximately 10000 to -1200 bp. The ubiquitous POU homeodomain protein Oct-1 recognized the A/T-rich silencer element at multiple sites in gel mobility shift assays and in vitro footprinting analyses. The silencing activity of Oct-1 was localized in its C-terminal alanine-rich domain, suggesting that Oct-1 plays a role in silencing of the hTSHbeta promoter. Further, a significant fraction of Oct-1 was shown to be associated with the nuclear matrix, and the hTSHbeta silencer region was tethered to a nuclear matrix of human cells in vivo, suggesting a possible role of the Oct-1-hTSHbeta silencer region interaction in chromatin organization.

AMPK Regulates Circadian Rhythms in a Tissue- and Isoform-Specific Manner

Background AMP protein kinase (AMPK) plays an important role in food intake and energy metabolism, which are synchronized to the light-dark cycle. In vitro, AMPK affects the circadian rhythm by regulating at least two clock components, CKIα and CRY1, via direct phosphorylation. However, it is not known whether the catalytic activity of AMPK actually regulates circadian rhythm in vivo. Methodology/Principal Finding The catalytic subunit of AMPK has two isoforms: α1 and α2. We investigate the circadian rhythm of behavior, physiology and gene expression in AMPKα1−/− and AMPKα2−/− mice. We found that both α1−/− and α2−/− mice are able to maintain a circadian rhythm of activity in dark-dark (DD) cycle, but α1−/− mice have a shorter circadian period whereas α2−/− mice showed a tendency toward a slightly longer circadian period. Furthermore, the circadian rhythm of body temperature was dampened in α1−/− mice, but not in α2−/− mice. The circadian pattern of core clock gene expression was severely disrupted in fat in α1−/− mice, but it was severely disrupted in the heart and skeletal muscle of α2−/− mice. Interestingly, other genes that showed circadian pattern of expression were dysreguated in both α1−/− and α2−/− mice. The circadian rhythm of nicotinamide phosphoryl-transferase (NAMPT) activity, which converts nicotinamide (NAM) to NAD+, is an important regulator of the circadian clock. We found that the NAMPT rhythm was absent in AMPK-deficient tissues and cells. Conclusion/Significance This study demonstrates that the catalytic activity of AMPK regulates circadian rhythm of behavior, energy metabolism and gene expression in isoform- and tissue-specific manners.

hnRNP U Inhibits Carboxy-Terminal Domain Phosphorylation by TFIIH and Represses RNA Polymerase II Elongation

ABSTRACT This study describes a potential new function of hnRNP U as an RNA polymerase (Pol II) elongation inhibitor. We demonstrated that a subfraction of human hnRNP U is associated with the Pol II holoenzyme in vivo and as such recruited to the promoter as part of the preinitiation complex. hnRNP U, however, appears to dissociate from the Pol II complex at the early stage of transcription and is therefore absent from the elongating Pol II complex. When tested in the human immunodeficiency virus type 1 transcription system, hnRNP U inhibits elongation rather than initiation of transcription by Pol II. This inhibition requires the carboxy-terminal domain (CTD) of Pol II. We showed that hnRNP U can bind TFIIH in vivo under certain conditions and inhibit TFIIH-mediated CTD phosphorylation in vitro. We find that the middle domain of hnRNP U is sufficient to mediate its Pol II association and its inhibition of TFIIH-mediated phosphorylation and Pol II elongation. The abilities of hnRNP U to inhibit TFIIH-mediated CTD phosphorylation and its Pol II association are necessary for hnRNP U to mediate the repression of Pol II elongation. Based on these observations, we suggest that a subfraction of hnRNP U, as a component of the Pol II holoenzyme, may downregulate TFIIH-mediated CTD phosphorylation in the basal transcription machinery and repress Pol II elongation. With such functions, hnRNP U might provide one of the mechanisms by which the CTD is maintained in an unphosphorylated state in the Pol II holoenzyme.

Immunopathology of Ocular Onchocerciasis: 2. Anti-retinal Autoantibodies in Serum and Ocular Fluids

Ocular fluids and sera from 12 onchocerciasis patients and nine age-matched controls living in Tamale, Ghana, were examined for the presence of anti-retinal autoantibodies by the indirect immunoperoxidase technique. Antibodies directed against autoantigens of the inner retina (nerve fiber, ganglion cell, and Müller cell) were found in the sera of 10 of 12 patients, but in only three of nine controls (P less than 0.003). Autoimmune antibodies against the outer segment of the photoreceptor were noted in 7 of 12 patients, in contrast to only one of nine controls (P less than 0.02). Findings with the ocular fluids generally mirrored those with the sera. These autoantibodies could not be absorbed by conventional techniques using either retinal S-antigen (S-Ag) or the interphotoreceptor retinoid binding protein, an observation suggesting that other ocular antigens are involved. Such anti-retinal antibodies, especially those directed against the inner retina, may play a significant role in the pathogenesis of the retinal degeneration and optic atrophy that occur as a consequence of onchocerciasis.

Promyelocytic Leukemia Activates Chk2 by Mediating Chk2 Autophosphorylation

Chk2 is a kinase critical for DNA damage-induced apoptosis and is considered a tumor suppressor. Chk2 is essential for p53 transcriptional and apoptotic activities. Although mutations of p53 are present in more than half of all tumors, mutations of Chk2 in cancers are rare, suggesting that Chk2 may be inactivated by unknown alternative mechanisms. Here we elucidate one such alternative mechanism regulated by PML (promyelocytic leukemia) that is involved in acute promyelocytic leukemia (APL). Although p53-inactivating mutations are extremely rare in APL, t(15;17) chromosomal translocation which fuses retinoic acid receptor (RARα) to PML is almost always present in APL, while the other PML allele is intact. We demonstrate that PML interacts with Chk2 and activates Chk2 by mediating its autophosphorylation step, an essential step for Chk2 activity that occurs after phosphorylation by the upstream kinase ATM (ataxia telangiectasia-mutated). PML/RARα in APL suppresses Chk2 by dominantly inhibiting the auto-phosphorylation step, but inactivation of PML/RARα with alltrans retinoic acid (ATRA) restores Chk2 autophosphorylation and activity. Thus, by fusing PML with RARα, the APL cells appear to have achieved functional suppression of Chk2 compromising the Chk2-p53 apoptotic pathway.