Purinergic glio-endothelial coupling during neuronal activity: role of P2Y1 receptors and eNOS in functional hyperemia in the mouse somatosensory cortex.

Impairment of moment-to-moment adjustment of cerebral blood flow (CBF) via neurovascular coupling is thought to play a critical role in the genesis of cognitive impairment associated with aging and pathological conditions associated with accelerated cerebromicrovascular aging (e.g., hypertension, obesity). Although previous studies demonstrate that endothelial dysfunction plays a critical role in neurovascular uncoupling in these conditions, the role of endothelial NO mediation in neurovascular coupling responses is not well understood. To establish the link between endothelial function and functional hyperemia, neurovascular coupling responses were studied in mutant mice overexpressing or deficient in endothelial NO synthase (eNOS), and the role of P2Y1 receptors in purinergic glioendothelial coupling was assessed. We found that genetic depletion of eNOS (eNOS(-/-)) and pharmacological inhibition of NO synthesis significantly decreased the CBF responses in the somatosensory cortex evoked by whisker stimulation and by administration of ATP. Overexpression of eNOS enhanced NO mediation of functional hyperemia. In control mice, the selective and potent P2Y1 receptor antagonist MRS2179 attenuated both whisker stimulation-induced and ATP-mediated CBF responses, whereas, in eNOS(-/-) mice, the inhibitory effects of MRS2179 were blunted. Collectively, our findings provide additional evidence for purinergic glio-endothelial coupling during neuronal activity, highlighting the role of ATP-mediated activation of eNOS via P2Y1 receptors in functional hyperemia.

Resveratrol increases cerebral glycogen synthase kinase phosphorylation as well as protein levels of drebrin and transthyretin in mice: an exploratory study.

Alzheimer's disease (AD) is characterized by intraneuronal ß-amyloid plaques and hyperphosphorylated tau, leading to neuronal cell death and progressive memory losses. This exploratory work investigates if dietary resveratrol, previously shown to have broad anti-aging effects and improve AD pathology in vivo, leads to neuroprotective changes in specific protein targets in the mouse brain. Both wild-type and APP/PS1 mice, a transgenic AD mouse model, received control AIN-93G diet or AIN-93G supplemented with resveratrol. Pathology parameters and AD risk were assessed via measurements on plaque burden, levels of phosphorylated glycogen synthase kinase 3-ß (GSK3-ß), tau, transthyretin and drebrin. Dietary resveratrol treatment did not decrease plaque burden in APP/PS1 mice. However, resveratrol-fed mice demonstrated increases in GSK3-ß phosphorylation, a 3.8-fold increase in protein levels of transthyretin, and a 2.2-fold increase in drebrin. This study broadens our understanding of specific mechanisms and targets whereby resveratrol provides neuroprotection.

Rapamycin has a biphasic effect on insulin sensitivity in C2C12 myotubes due to sequential disruption of mTORC1 and mTORC2.

Rapamycin, an inhibitor of mTOR complex 1 (mTORC1), improves insulin sensitivity in acute studies in vitro and in vivo by disrupting a negative feedback loop mediated by S6 kinase. We find that rapamycin has a clear biphasic effect on insulin sensitivity in C2C12 myotubes, with enhanced responsiveness during the first hour that declines to almost complete insulin resistance by 24-48 h. We and others have recently observed that chronic rapamycin treatment induces insulin resistance in rodents, at least in part due to disruption of mTORC2, an mTOR-containing complex that is not acutely sensitive to the drug. Chronic rapamycin treatment may also impair insulin action via the inhibition of mTORC1-dependent mitochondrial biogenesis and activity, which could result in a buildup of lipid intermediates that are known to trigger insulin resistance. We confirmed that rapamycin inhibits expression of PGC-1α, a key mitochondrial transcription factor, and acutely reduces respiration rate in myotubes. However, rapamycin did not stimulate phosphorylation of PKCθ, a central mediator of lipid-induced insulin resistance. Instead, we found dramatic disruption of mTORC2, which coincided with the onset of insulin resistance. Selective inhibition of mTORC1 or mTORC2 by shRNA-mediated knockdown of specific components (Raptor and Rictor, respectively) confirmed that mitochondrial effects of rapamycin are mTORC1-dependent, whereas insulin resistance was recapitulated only by knockdown of mTORC2. Thus, mTORC2 disruption, rather than inhibition of mitochondria, causes insulin resistance in rapamycin-treated myotubes, and this system may serve as a useful model to understand the effects of rapamycin on mTOR signaling in vivo.

SIRT1 is required for AMPK activation and the beneficial effects of resveratrol on mitochondrial function.

Resveratrol induces mitochondrial biogenesis and protects against metabolic decline, but whether SIRT1 mediates these benefits is the subject of debate. To circumvent the developmental defects of germline SIRT1 knockouts, we have developed an inducible system that permits whole-body deletion of SIRT1 in adult mice. Mice treated with a moderate dose of resveratrol showed increased mitochondrial biogenesis and function, AMPK activation, and increased NAD(+) levels in skeletal muscle, whereas SIRT1 knockouts displayed none of these benefits. A mouse overexpressing SIRT1 mimicked these effects. A high dose of resveratrol activated AMPK in a SIRT1-independent manner, demonstrating that resveratrol dosage is a critical factor. Importantly, at both doses of resveratrol no improvements in mitochondrial function were observed in animals lacking SIRT1. Together these data indicate that SIRT1 plays an essential role in the ability of moderate doses of resveratrol to stimulate AMPK and improve mitochondrial function both in vitro and in vivo.

Mammalian DNA δ15N exhibits 40‰ intramolecular variation and is unresponsive to dietary protein level.

We report the first high-precision characterization of molecular and intramolecular δ(15)N of nucleosides derived from mammalian DNA. The influence of dietary protein level on brain amino acids and deoxyribonucleosides was determined to investigate whether high protein turnover would alter amino acid (15)N or (13)C values. Pregnant guinea pig dams were fed control diets, or high or low levels of dietary protein throughout gestation, and all pups were fed control diets. The cerebellar DNA of offspring was extracted at 2 and 120 days of life, nucleosides isolated and δ(15)N and δ(13)C values characterized. Mean diet δ(15)N was 0.45 ± 0.33‰, compared with cerebellar whole tissue and DNA δ(15) N= +4.1 ± 0.7‰ and -4.5 ± 0.4‰, respectively. Cerebellar deoxythymidine (dT), deoxycytidine (dC), deoxyadenosine (dA), and deoxyguanosine (dG) δ(15)N were +1.4 ± 0.4, -2.1 ± 0.9, -7.2 ± 0.3, and -10.4 ± 0.5‰, respectively. There were no changes in amino acid or deoxyribonucleoside δ(15) N values due to dietary protein level. Using known metabolic relationships, we developed equations to calculate the intramolecular δ(15)N values originating from aspartate (asp) in purines (pur) or pyrimidines (pyr), glutamine (glu), and glycine (gly) to be δ(15)N(ASP-PUR), δ(15)N(ASP-PYR), δ(15) N(GLN), and δ(15) N(GLY) +11.9 ± 2.3‰, +7.0 ± 2.0‰, -9.1 ± 2.4‰, and -31.8 ± 8.9‰, respectively. A subset of twelve amino acids from food and brain had mean δ(15) N values of 4.3 ± 3.2‰ and 13.8 ± 3.1‰, respectively, and δ(15)N values for gly and asp were 12.6 ± 2.2‰ and 15.2 ± 0.8‰, respectively. A separate isotope tracer study detected no significant turnover of cerebellar DNA in the first six months of life. The large negative δ(15)N difference between gly and cerebellar purine N at the gly (7) position implies either that there is a major isotope effect during DNA synthesis, or that in utero gly has a different isotope ratio during rapid growth and metabolism from that in adult life. Our data show that cerebellar nucleoside intramolecular δ(15)N values vary over more than 40‰ and are not influenced by dietary protein level or age.

Roles of hormones and signaling molecules in describing the relationship between obesity and colon cancer.

Colon cancer represents a highly prevalent disease in the Western world. While dietary and lifestyle recommendations remain important factors in disease prevention and treatment, epidemiological data have made it clear that obesity and excess body weight remain significant risk factors for the disease. A number of potential direct and indirect relationships exist between obesity and increased risk of colon cancer. Several mechanisms which appear promising and warrant further investigation are discussed here, specifically the modifying role of insulin and insulin-like growth factors, leptin, adipose-tissue induced changes in estrogens and androgens, and inflammatory molecules. A brief review of these hormones and signaling molecules and their action in colon cancer development is described. A thorough integration and understanding of the mechanisms of action these systems exert on colonic epithelia will be important in designing studies and experiments aimed at elucidating disease etiology for prevention and treatment.

Docosahexaenoic and arachidonic acid concentrations in human breast milk worldwide.

Concentrations of the long-chain polyunsaturated fatty acids (LCPUFAs) docosahexaenoic acid (DHA, 22:6n-3) and arachidonic acid (AA, 20:4n-6) in human breast milk are important indicators of infant formula DHA and AA concentrations, and recent evidence suggests that neural maturation of breastfed infants is linked to breast-milk LCPUFA concentrations. We report a descriptive meta-analysis that considered 106 studies of human breast milk culled to include only studies that used modern analysis methods capable of making accurate estimates of fatty acid (FA) profiles and criteria related to the completeness of reporting. The final analysis included 65 studies of 2474 women. The mean (+/-SD) concentration of DHA in breast milk (by wt) is 0.32 +/- 0.22% (range: 0.06-1.4%) and that of AA is 0.47 +/- 0.13% (range: 0.24-1.0%), which indicates that the DHA concentration in breast milk is lower than and more variable than that of AA. The highest DHA concentrations were primarily in coastal populations and were associated with marine food consumption. The correlation between breast-milk DHA and AA concentrations was significant but low (r = 0.25, P = 0.02), which indicates that the mean ratio of DHA to AA in regional breast milk varies widely. This comprehensive analysis of breast-milk DHA and AA indicates a broad range of these nutrients worldwide and serves as a guide for infant feeding.