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 CamKKß-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.

Acquired Phototrophy as an Evolutionary Path to Mixotrophy.

AbstractAcquired photosynthesis transforms genotypically heterotrophic lineages into autotrophs. Transient acquisitions of eukaryotic chloroplasts may provide key evolutionary insight into the endosymbiosis process-the hypothesized mechanism by which eukaryotic cells obtained new functions via organelle retention. Here, we use an eco-evolutionary model to study the environmental conditions under which chloroplast retention is evolutionarily favorable. We focus on kleptoplastidic lineages-which steal functional chloroplasts from their prey-as hypothetical evolutionary intermediates. Our adaptive dynamics analysis reveals a spectrum of evolutionarily stable strategies ranging from phagotrophy to phototrophy to obligate kleptoplasty. Our model suggests that a low-light niche and weak (or absent) trade-offs between chloroplast retention and overall digestive ability favor the evolution of phototrophy. In contrast, when consumers experience strong trade-offs, obligate kleptoplasty emerges as an evolutionary end point. Therefore, the preevolved trade-offs that underlie an evolving lineage's physiology will likely constrain its evolutionary trajectory.

Fertilization by coral-dwelling fish promotes coral growth but can exacerbate bleaching response.

Many corals form close associations with a diverse assortment of coral-dwelling fishes and other fauna. As coral reefs around the world are increasingly threatened by mass bleaching events, it is important to understand how these biotic interactions influence corals' susceptibility to bleaching. We used dynamic energy budget modeling to explore how nitrogen excreted by coral-dwelling fish affects the physiological performance of host corals. In our model, fish presence influenced the functioning of the coral-Symbiodiniaceae symbiosis by altering nitrogen availability, and the magnitude and sign of these effects depended on environmental conditions. Although our model predicted that fish-derived nitrogen can promote coral growth, the relationship between fish presence and coral tolerance of photo-oxidative stress was non-linear. Fish excretions supported denser symbiont populations that provided protection from incident light through self-shading. However, these symbionts also used more of their photosynthetic products for their own growth, rather than sharing with the coral host, putting the coral holobiont at a higher risk of becoming carbon-limited and bleaching. The balance between the benefits of increased symbiont shading and costs of reduced carbon sharing depended on environmental conditions. Thus, while there were some scenarios under which fish presence increased corals' tolerance of light stress, fish could also exacerbate bleaching and slow or prevent subsequent recovery. We discuss how the contrast between the potentially harmful effects of fish predicted by our model and results of empirical studies may relate to key model assumptions that warrant further investigation. Overall, this study provides a foundation for future work on how coral-associated fauna influence the bioenergetics of their host corals, which in turn has implications for how these corals respond to bleaching-inducing stressors.

Metabolic sensor AMPK directly phosphorylates RAG1 protein and regulates V(D)J recombination.

The ability to sense metabolic stress is critical for successful cellular adaptation. In eukaryotes, the AMP-activated protein kinase (AMPK), a highly conserved serine/threonine kinase, functions as a critical metabolic sensor. AMPK is activated by the rising ADP/ATP and AMP/ATP ratios during conditions of energy depletion and also by increasing intracellular Ca(2+). In response to metabolic stress, AMPK maintains energy homeostasis by phosphorylating and regulating proteins that are involved in many physiological processes including glucose and fatty acid metabolism, transcription, cell growth, mitochondrial biogenesis, and autophagy. Evidence is mounting that AMPK also plays a role in a number of pathways unrelated to energy metabolism. Here, we identify the recombination-activating gene 1 protein (RAG1) as a substrate of AMPK. The RAG1/RAG2 complex is a lymphoid-specific endonuclease that catalyzes specific DNA cleavage during V(D)J recombination, which is required for the assembly of the Ig and T-cell receptor genes of the immune system. AMPK directly phosphorylates RAG1 at serine 528, and the phosphorylation enhances the catalytic activity of the RAG complex, resulting in increased cleavage of oligonucleotide substrates in vitro, or increased recombination of an extrachromosomal substrate in a cellular assay. Our results suggest that V(D)J recombination can be regulated by AMPK activation, providing a potential new link between metabolic stress and development of B and T lymphocytes.

Upregulation of brain-derived neurotrophic factor expression in nodose ganglia and the lower brainstem of hypertensive rats.

Hypertension leads to structural and functional changes at baroreceptor synapses in the medial nucleus tractus solitarius (NTS), but the underlying molecular mechanisms remain unknown. Our previous studies show that brain-derived neurotrophic factor (BDNF) is abundantly expressed by rat nodose ganglion (NG) neurons, including baroreceptor afferents and their central terminals in the medial NTS. We hypothesized that hypertension leads to upregulation of BDNF expression in NG neurons. To test this hypothesis, we used two mechanistically distinct models of hypertension, the spontaneously hypertensive rat (SHR) and the deoxycorticosterone acetate (DOCA)-salt rat. Young adult SHRs, whose blood pressure was significantly elevated compared with age-matched Wistar-Kyoto (WKY) control rats, exhibited dramatic upregulation of BDNF mRNA and protein in the NG. BDNF transcripts from exon 4, known to be regulated by activity, and exon 9 (protein-coding region) showed the largest increases. Electrical stimulation of dispersed NG neurons with patterns that mimic baroreceptor activity during blood pressure elevations led to increases in BDNF mRNA that were also mediated through promoter 4. The increase in BDNF content of the NG in vivo was associated with a significant increase in the percentage of BDNF-immunoreactive NG neurons. Moreover, upregulation of BDNF in cell bodies of NG neurons was accompanied by a significant increase in BDNF in the NTS region, the primary central target of NG afferents. A dramatic increase in BDNF in the NG was also detected in DOCA-salt hypertensive rats. Together, our study identifies BDNF as a candidate molecular mediator of activity-dependent changes at baroafferent synapses during hypertension.

Percutaneous core biopsy of small renal mass lesions: a diagnostic tool to better stratify patients for surgical intervention.

OBJECTIVES: To further validate the safety and diagnostic accuracy of percutaneous core biopsy in small renal masses ([SRMs]≤4 cm) in response to the rising prevalence of renal 'incidentalomas'. To determine the value of percutaneous core biopsy in its ability to influence the choice of intervention or surveillance for the management of SRMs. PATIENTS AND METHODS: We collected data on the incidence of benign, malignant and non-diagnostic samples from 268 SRM (clinical T1a) biopsies performed at our institution between 1998 and 2009. The diagnostic accuracy of biopsy in small renal lesions was examined in cases proceeding to nephrectomy. Follow-up on the remaining non-surgical cases was performed. RESULTS: SRMs (≤4 cm, clinical T1a) constituted 59% of all renal lesions diagnosed; 80% of these biopsies were diagnostic and 20% were non-diagnostic. Of the diagnostic samples, 74% were malignant and 26% were benign. Thirty-three percent (17 of the initial 52) non-diagnostic samples underwent re-biopsy, with 94% yielding a histological diagnosis on repeat sampling. Fifty-eight percent of our diagnostic masses underwent nephrectomy, returning a renal biopsy accuracy rate of 100% for identifying lesions as benign or malignant. Of the 98 observed masses, 49% were benign, 28% malignant and 23% were non-diagnostic. In 63% of these cases, size remained stable in the follow-up period. CONCLUSIONS: Percutaneous core biopsy continues to provide an accurate and safe tool for preoperative tissue diagnosis of SRMs and should be offered to patients before considering surgical intervention. With regard to both benign and malignant T1a renal lesions, in select patient groups, those electing to undergo observation may expect favourable short- to medium-term outcomes.

Inverted papilloma of the urinary tract.

OBJECTIVES: • To compare the clinical and pathological details of inverted papilloma (IP) of the urinary tract diagnosed in Western Australia with those published. • To determine whether urinary tract IP requires post-treatment cystoscopic follow-up. PATIENTS AND METHODS: • Clinical and pathological details were summarized for 41 cases of IP of the urinary tract diagnosed in Western Australia between 1998 and 2010. • Publications on IP of the urinary tract were reviewed and summarized. RESULTS: • IP of the urinary tract is a rare benign tumour most commonly diagnosed in older men presenting with haematuria or symptoms of lower urinary tract obstruction. • IP is most frequently identified in the bladder neck or trigone as a polypoid growth with a smooth surface. • The major differential diagnosis is transitional cell carcinoma (TCC) with an inverted growth pattern, with differentiation based mainly on morphological criteria. • Treatment involves transurethral resection for lower urinary tract lesions whereas upper urinary tract IPs are resected by ureteroscopy, percutaneous endoscopy, partial ureterectomy or nephroureterectomy. • IP is weakly associated with a history of TCC and with increased risk of concomitant or subsequent TCC. CONCLUSIONS: • Based on the association with TCC, post-treatment follow-up for IP of the urinary tract should include cystoscopic follow-up.

Baseline Results: The Association Between Cardiovascular Risk and Preclinical Alzheimer's Disease Pathology (ASCEND) Study.

BACKGROUND: The rate of AD for African Americans (AAs) is 64% higher than for non-Hispanic White Americans (Whites). It is hypothesized that poor peripheral vascular function, in combination with genetics, stress, and inflammation may directly contribute to the accumulation of AD pathologic biomarkers. These risk factors may disproportionately affect AAs. OBJECTIVE: Our objective was to determine if in a healthy middle-aged cohort at risk for AD (1) AD biomarkers in CSF differ by race, (2) peripheral vascular dysfunction and cognition are related to a higher burden of CSF AD biomarkers, and (3) these relationships differ by race. METHODS: We enrolled 82 cognitively normal, middle-aged (45 and older) adults including AAs and Whites at high risk for AD due to parental history. Study procedures included lumbar puncture, vascular ultrasound, and cognitive testing. RESULTS: While participants were in overall good health, AAs exhibited poorer indices of preclinical vascular health, including higher central SBP, central MAP, and EndoPAT AI, a marker of arterial stiffness. AAs also had significantly less cerebrospinal fluid tau burden than Whites. After polynomial regression analysis, adjusted for age, gender, education, and ApoE4 status, race significantly modified the relationship between total tau, phospho-tau, and Trails B, a marker of executive function. Small differences in tau correlated with poorer cognition in AAs. CONCLUSION: In a healthy middle-aged cohort at risk for AD, AAs had worse peripheral vascular health and worse cognition than Whites. Despite lower tau burden overall, race modified the relationship between tau and cognition, such that small differences in tau between AAs was related to worse cognition when compared to Whites.

VDAC oligomers form mitochondrial pores to release mtDNA fragments and promote lupus-like disease.

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.

Rationale and Design of the Mechanistic Potential of Antihypertensives in Preclinical Alzheimer's (HEART) Trial.

Research indicates that certain antihypertensive medications alter Alzheimer's disease (AD) biomarkers in Caucasians. The renin angiotensin system (RAS) regulates blood pressure (BP) in the body and the brain and may directly influence AD biomarkers, including amyloid-ß (Aß) neuropathology, cerebral blood flow (CBF), and inflammatory markers. This hypothesis is supported by studies, including ours, showing that antihypertensives targeting the RAS reduce the risk and slow the progression of AD in Caucasians. While mounting evidence supports a protective role of RAS medications in Caucasians, this mechanism has not been explored in African Americans. To assess the mechanism by which RAS medications modify the brain RAS, cerebrospinal fluid (CSF) Aß, CBF, and inflammatory markers in African Americans, we are conducting an eight month, Phase Ib randomized, placebo controlled trial, enrolling 60 middle-aged (45-70 years), non-demented individuals, at risk for AD by virtue of a parental history. Participants include normotensive and treated hypertensives that have never been exposed to a RAS medication. Participants are randomized (1 : 1:1) by gender and BP medication use (yes/no) to one of three groups: placebo, or 20 mg, or 40 mg telmisartan (Micardis), to determine the dose required to penetrate the CNS. Our overarching hypothesis is that, compared to placebo, both doses of telmisartan will penetrate the CNS and produce salutary, dose dependent effects on the brain RAS as well as CSF Aß, CBF, and CSF inflammatory markers in African Americans, over eight months. This manuscript describes the trial rationale and design.

Identification of novel substrates for human checkpoint kinase Chk1 and Chk2 through genome-wide screening using a consensus Chk phosphorylation motif.

Checkpoint kinase 1 (Chk1) and Chk2 are effector kinases in the cellular DNA damage response and impairment of their function is closely related to tumorigenesis. Previous studies revealed several substrate proteins of Chk1 and Chk2, but identification of additional targets is still important in order to understand their tumor suppressor functions. In this study, we screened novel substrates for Chk1 and Chk2 using substrate target motifs determined previously by an oriented peptide library approach. The potential candidates were selected by genome-wide peptide database searches and were examined by in vitro kinase assays. ST5, HDAC5, PGC-1alpha, PP2A PR130, FANCG, GATA3, cyclin G, Rad51D and MAD1a were newly identified as in vitro substrates for Chk1 and/or Chk2. Among these, HDAC5 and PGC-1a were further analyzed to substantiate the screening results. Immunoprecipitation kinase assay of full-length proteins and site-directed mutagenesis analysis of the target motifs demonstrated that HDAC5 and PGC-1alpha were specific targets for Chk1 and/or Chk2 at least in vitro.

Prenatal choline supplementation attenuates MK-801-induced deficits in memory, motor function, and hippocampal plasticity in adult male rats.

Choline is essential to the development and function of the central nervous system and supplemental choline during development is neuroprotective against a variety of insults, including neurotoxins like dizocilpine (MK-801). MK-801 is an NMDA receptor antagonist that is frequently used in rodent models of psychological disorders, particularly schizophrenia. At low doses, it causes cognitive impairments, and at higher doses it induces motor deficits, anhedonia, and neuronal degeneration. The primary goals of the present study were to investigate whether prenatal choline supplementation protects against the cognitive impairments, motor deficits, and neuropathologies that are precipitated by MK-801 administration in adulthood. Adult male Sprague-Dawley rats were fed a standard or supplemented choline diet prenatally. Using the novelty preference test of object recognition, we found that only prenatal standard-fed rats displayed memory consolidation deficits induced by low-dose MK-801 administered immediately following study of sample objects; all other groups, including prenatal choline supplemented rats given MK-801, showed intact memory. Following high-dose MK-801, prenatal choline supplementation significantly alleviated rats' motor response to MK-801, particularly ataxia. Using doublecortin and Ki67 to mark neurogenesis and cell division, respectively, in the hippocampus, we found that prenatal choline supplementation, in the face of MK-801 toxicity, protected against reduced hippocampal plasticity. Taken together, the current findings suggest that prenatal choline supplementation protects against a variety of behavioral and neural pathologies induced by the neurotoxin, MK-801. This research contributes to the growing body of evidence supporting the robust neuroprotective capacity of choline.

Specific Sirt1 Activator-mediated Improvement in Glucose Homeostasis Requires Sirt1-Independent Activation of AMPK.

The specific Sirt1 activator SRT1720 increases mitochondrial function in skeletal muscle, presumably by activating Sirt1. However, Sirt1 gain of function does not increase mitochondrial function, which raises a question about the central role of Sirt1 in SRT1720 action. Moreover, it is believed that the metabolic effects of SRT1720 occur independently of AMP-activated protein kinase (AMPK), an important metabolic regulator that increases mitochondrial function. Here, we show that SRT1720 activates AMPK in a Sirt1-independent manner and SRT1720 activates AMPK by inhibiting a cAMP degrading phosphodiesterase (PDE) in a competitive manner. Inhibiting the cAMP effector protein Epac prevents SRT1720 from activating AMPK or Sirt1 in myotubes. Moreover, SRT1720 does not increase mitochondrial function or improve glucose tolerance in AMPKα2 knockout mice. Interestingly, weight loss induced by SRT1720 is not sufficient to improve glucose tolerance. Therefore, contrary to current belief, the metabolic effects produced by SRT1720 require AMPK, which can be activated independently of Sirt1.

DNA-PK Promotes the Mitochondrial, Metabolic, and Physical Decline that Occurs During Aging.

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 decline of physical fitness in middle-aged mice and protects against type 2 diabetes. In conclusion, DNA-PK is one of the drivers of the metabolic and fitness decline during aging, and therefore DNA-PK inhibitors may have therapeutic potential in obesity and low exercise capacity.

Dendritic cells induce Th2-mediated airway inflammatory responses to house dust mite via DNA-dependent protein kinase.

DNA-dependent protein kinase (DNA-PK) mediates double-stranded DNA break repair, V(D)J recombination and immunoglobulin class switch recombination, as well as innate immune and pro-inflammatory responses. However, there is limited information regarding the role of DNA-PK in adaptive immunity mediated by dendritic cells (DCs), which are the primary antigen-presenting cells in allergic asthma. Here we show that house dust mite induces DNA-PK phosphorylation, which is a marker of DNA-PK activation, in DCs via the generation of intracellular reactive oxygen species. We also demonstrate that pharmacological inhibition of DNA-PK, as well as the specific deletion of DNA-PK in DCs, attenuates the induction of allergic sensitization and Th2 immunity via a mechanism that involves the impaired presentation of mite antigens. Furthermore, pharmacological inhibition of DNA-PK following antigen priming similarly reduces the manifestations of mite-induced airway disease. Collectively, these findings suggest that DNA-PK may be a potential target for treatment of allergic asthma.

Oxygen tension regulates the stability of insulin receptor substrate-1 (IRS-1) through caspase-mediated cleavage.

The insulin and insulin-like growth factor-1 (IGF-1) receptors mediate signaling for energy uptake and growth through insulin receptor substrates (IRSs), which interact with these receptors as well as with downstream effectors. Oxygen is essential not only for ATP production through oxidative phosphorylation but also for many cellular processes, particularly those involved in energy homeostasis. The oxygen tension in vivo is significantly lower than that in the air and can vary widely depending on the tissue as well as on perfusion and oxygen consumption. How oxygen tension affects IRSs and their functions is poorly understood. Our findings indicate that transient hypoxia (1% oxygen) leads to caspase-mediated cleavage of IRS-1 without inducing cell death. The IRS-1 protein level rebounds rapidly upon return to normoxia. Protein tyrosine phosphatases (PTPs) appear to be important for the IRS-1 cleavage because tyrosine phosphorylation of the insulin receptor was decreased in hypoxia and IRS-1 cleavage could be blocked either with H(2)O(2) or with vanadate, each of which inhibits PTPs. Activity of Akt, a downstream effector of insulin and IGF-1 signaling that is known to suppress caspase activation, was suppressed in hypoxia. Overexpression of dominant-negative Akt led to IRS-1 cleavage even in normoxia, and overexpression of constitutively active Akt partially suppressed IRS-1 cleavage in hypoxia, suggesting that hypoxia-mediated suppression of Akt may induce caspase-mediated IRS-1 cleavage. In conclusion, our study elucidates a mechanism by which insulin and IGF-1 signaling can be matched to the oxygen level that is available to support growth and energy metabolism.

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 (RARalpha) 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/RARalpha in APL suppresses Chk2 by dominantly inhibiting the auto-phosphorylation step, but inactivation of PML/RARalpha with alltrans retinoic acid (ATRA) restores Chk2 autophosphorylation and activity. Thus, by fusing PML with RARalpha, the APL cells appear to have achieved functional suppression of Chk2 compromising the Chk2-p53 apoptotic pathway.