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.

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...

BackgroundAMP 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 FindingThe 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/SignificanceThis study demonstrates that the catalytic activity of AMPK regulates circadian rhythm of behavior, energy metabolism and gene expression in isoform- and tissue-specific manners.

SUMMARY Hallmarks of aging that negatively impact health include weight gain and reduced physical fitness, which can increase insulin resistance and risk for many diseases including type 2 diabetes. The underlying mechanism(s) for these phenomena is poorly understood. Here we report that aging increases DNA breaks and activates DNA-dependent protein kinase (DNA-PK) in skeletal muscle, which suppresses mitochondrial function, energy metabolism and physical fitness. DNA-PK phosphorylates threonines 5 and 7 of HSP90α, decreasing its chaperone function for clients such as AMP-activated protein kinase (AMPK), which is critical for mitochondrial biogenesis and energy metabolism. Decreasing DNA-PK activity increases AMPK activity and prevents weight gain, decline of mitochondrial function and physical fitness in middle aged mice and protects against type 2 diabetes. Therefore, DNA-PK is one of the drivers of the metabolic and fitness decline during aging, which make staying lean and physically fit difficult and increase susceptibility to metabolic diseases.

Abbreviations: AP1, activator protein 1; C/EBP-β, cAMP response element-binding protein β; DNA-PK, DNA-dependent protein kinase; Egr1, early growth response gene product 1; HIF-1, hypoxia inducible factor-1; NF-κB, nuclear factor kappa B A bstract

In this study, we investigated possible engagement of NF-kB and Ku autoantigen (Ku) activation in development of multidrug resistance (MDR) and circumvention of MDR by modulation of NF-kB and Ku. The NF-kB activity and NF-kB p65 subunit level were constitutively higher in MDR cells than in drug-sensitive parental cells. Interestingly, a faster running NF-kB DNA binding complex was identi®ed as Ku, a DNA damage sensor and a key double strand break repair protein, and was positively correlated with the NF-kB activity in MDR cells and Ku-or both subunits of NF-kB-transfected cells. Also both NF-kB and Ku activities were activated or inhibited by treatment with etoposide (VP-16) or MG-132 (a proteasome inhibitor), respectively. Furthermore, PKA inhibitor suppressed markedly the constitutive and drug-induced activities of NF-kB and Ku in MDR cells and subsequently potentiated the cytotoxic activity of anticancer drugs. Our results proposed that the NF-kB and Ku activation could be one of multi-factorial MDR mechanism, and PKA inhibitor, likely via inhibition of NF-kB and Ku activities, could enhance the e ectiveness of anticancer drugs against MDR cells with high activities of NF-kB and Ku. Oncogene (2001) 20, 6048 ± 6056.

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The failure to treat metastatic cancer with multidrug resistance is a major problem for successful cancer therapy, and the molecular basis for the association of metastatic phenotype with resistance to therapy is still unclear. In this study, we revealed that various metastatic cancer cells showed consistently higher levels of antiapoptotic proteins, including Bcl-2, nuclear factor-B, MDM2, DNA-dependent protein kinase (DNA-PK), and epidermal growth factor receptor (EGFR), and lower levels of proapoptotic proteins, including Bax and p53 than low metastatic parental cells. This was followed by chemo-and radioresistance in metastatic cancer cells compared with their parental cells. EGFR and DNA-PK activity, which are known to be associated with chemo-and radioresistance, were demonstrated to be mutually regulated by each other. Treatment with PKI166, an EGFR inhibitor, suppressed etoposide-induced activation of DNA-PK in A375SM metastatic melanoma cells. In addition, PKI166 enhanced markedly the chemosensitivities of metastatic cancer cell sublines to various anticancer drugs in comparison with those of low metastatic cancer cells. These results suggest that the activities of DNA-PK and EGFR, which is positively correlated with each other, may contribute to metastatic phenotype as well as therapy resistance, and the EGFR inhibitor enhances the effect of anticancer drugs against therapy-resistant metastatic cancer cells via suppression of stress responses, including activation of DNA-PK.