SIRT1 Overexpression Antagonizes Cellular Senescence with Activated ERK/S6k1 Signaling in Human Diploid Fibroblasts

Sir2, a NAD-dependent deacetylase, modulates lifespan in yeasts, worms and flies. The SIRT1, mammalian homologue of Sir2, regulates signaling for favoring survival in stress. But whether SIRT1 has the function to influence cell viability and senescence under non-stressed conditions in human diploid fibroblasts is far from unknown. Our data showed that enforced SIRT1 expression promoted cell proliferation and antagonized cellular senescence with the characteristic features of delayed Senescence-Associated β-galactosidase (SA-β-gal) staining, reduced Senescence-Associated Heterochromatic Foci (SAHF) formation and G1 phase arrest, increased cell growth rate and extended cellular lifespan in human fibroblasts, while dominant-negative SIRT1 allele (H363Y) did not significantly affect cell growth and senescence but displayed a bit decreased lifespan.. Western blot results showed that SIRT1 reduced the expression of p16INK4A and promoted phosphorylation of Rb. Our data also exposed that overexpression of SIRT1 was accompanied by enhanced activation of ERK and S6K1 signaling. These effects were mimicked in both WI38 cells and 2BS cells by concentration-dependent resveratrol, a SIRT1 activator. It was noted that treatment of SIRT1-.transfected cells with Rapamycin, a mTOR inhibitor, reduced the phosphorylation of S6K1 and the expression of Id1, implying that SIRT1-induced phosphorylation of S6K1 may be partly for the decreased expression of p16INK4A and promoted phosphorylation of Rb in 2BS. It was also observed that the expression of SIRT1 and phosphorylation of ERK and S6K1 was declined in senescent 2BS. These findings suggested that SIRT1-promoted cell proliferation and antagonized cellular senescence in human diploid fibroblasts may be, in part, via the activation of ERK/ S6K1 signaling

Barcoding a Quantified Food Web: Crypsis, Concepts, Ecology and Hypotheses

The efficient and effective monitoring of individuals and populations is critically dependent on correct species identification. While this point may seem obvious, identifying the majority of the more than 100 natural enemies involved in the spruce budworm (Choristoneura fumiferana – SBW) food web remains a non-trivial endeavor. Insect parasitoids play a major role in the processes governing the population dynamics of SBW throughout eastern North America. However, these species are at the leading edge of the taxonomic impediment and integrating standardized identification capacity into existing field programs would provide clear benefits. We asked to what extent DNA barcoding the SBW food web would alter our understanding of the diversity and connectence of the food web and the frequency of generalists vs. specialists in different forest habitats. We DNA barcoded over 10% of the insects collected from the SBW food web in three New Brunswick forest plots from 1983 to 1993. For 30% of these specimens, we amplified at least one additional nuclear region. When the nodes of the food web were estimated based on barcode divergences (using molecular operational taxonomic units (MOTU) or phylogenetic diversity (PD) – the food web became much more diverse and connectence was reduced. We tested one measure of food web structure (the “bird feeder effect”) and found no difference compared to the morphologically based predictions. Many, but not all, of the presumably polyphagous parasitoids now appear to be morphologically-cryptic host-specialists. To our knowledge, this project is the first to barcode a food web in which interactions have already been well-documented and described in space, time and abundance. It is poised to be a system in which field-based methods permit the identification capacity required by forestry scientists. Food web barcoding provided an effective tool for the accurate identification of all species involved in the cascading effects of future budworm outbreaks. Integrating standardized barcodes within food webs may ultimately change the face of community ecology. This will be most poignantly felt in food webs that have not yet been quantified. Here, more accurate and precise connections will be within the grasp of any researcher for the first time

THE RELATIONSHIP AMONG PHYSICAL ACTIVITY LEVEL, ACTIVITY TYPE, AND THERMAL PAIN SENSITIVITY

Natalie R. Janzen, Jamie K. Huber, Carl J. Ade, E. Laurette Taylor, and Christopher D. Black. Department of Health and Exercise Science, University of Oklahoma, Norman, OK; e-mail: [email protected] Pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage. Previous research has demonstrated a potential relationship between pain sensitivity and total daily physical activity. However, no study has examined whether the type of physical activity may also influence pain sensitivity. PURPOSE: The purpose of this study was to examine differences in thermal pain sensitivity among college-aged females who engage in different amounts and types of physically activity. METHODS: Participants (n=27) were tested on two occasions. For the first visit participants went through informed consent, completed a menstrual history questionnaire, and were then familiarized with the protocol for pain sensitivity testing. Participants then wore an accelerometer at the waist for 7 days during waking hours (excluding water activity). The second visit was timed to occur during luteal phase of their menstrual cycle. Pain sensitivity was determined by having participants provide ratings of pain intensity (PI; 0-20) and ratings of pain unpleasantness (PU; 0-20) in response to brief (15-sec) applications of temperatures ranging from 43-49° C. Based upon their self-reported and measured activity levels and activity types participants were placed into the following groups: aerobically trained (AERO), resistance trained (RES), aerobic and resistance trained (A+R), and sedentary (SED). RESULTS: Total activity differed among the groups with the AERO (203±83 min) and A+R (183±28 min) groups accumulating more physical activity compared to the RES (39±39 min; p\u3c0.05) and SED (62±32 min; p\u3c0.05) groups. AERO (32±13 min) and A+R (34±7 min) also accumulated greater “vigorous” intensity activity than the RES (10±4 min; p\u3c0.05) and SED (12±5 min) groups. Mean ratings of PI across all temperatures did not differ among activity groups for (6.0±3.2, 7.4±2.9, 6.6±3.5, and 5.3±3.3 for AERO, RES, A+R, and SED, respectively; p=0.652) nor did ratings of PU across all temperatures (4.1±2.3, 4.6±2.5. 4.5±3.0, and 3.5±2.4 for AERO, RES, A+R, and SED, respectively; p=0.78). CONCLUSIONS: Unlike previous results in middle-aged and older women where higher activity was associated with lower pain sensitivity, our results suggest pain sensitivity does not differ among individuals with differing activity levels in college-aged females. Additionally, we demonstrated pain sensitivity also did not differ among differing types of physical activity

Genetic loss of AMPK-glycogen binding destabilises AMPK and disrupts metabolism

OBJECTIVE: Glycogen is a major energy reserve in liver and skeletal muscle. The master metabolic regulator AMP-activated protein kinase (AMPK) associates with glycogen via its regulatory β subunit carbohydrate-binding module (CBM). However, the physiological role of AMPK-glycogen binding in energy homeostasis has not been investigated in vivo. This study aimed to determine the physiological consequences of disrupting AMPK-glycogen interactions. METHODS: Glycogen binding was disrupted in mice via whole-body knock-in (KI) mutation of either the AMPK β1 (W100A) or β2 (W98A) isoform CBM. Systematic whole-body, tissue and molecular phenotyping was performed in KI and respective wild-type (WT) mice. RESULTS: While β1 W100A KI did not affect whole-body metabolism or exercise capacity, β2 W98A KI mice displayed increased adiposity and impairments in whole-body glucose handling and maximal exercise capacity relative to WT. These KI mutations resulted in reduced total AMPK protein and kinase activity in liver and skeletal muscle of β1 W100A and β2 W98A, respectively, versus WT mice. β1 W100A mice also displayed loss of fasting-induced liver AMPK total and α-specific kinase activation relative to WT. Destabilisation of AMPK was associated with increased fat deposition in β1 W100A liver and β2 W98A skeletal muscle versus WT. CONCLUSIONS: These results demonstrate that glycogen binding plays critical roles in stabilising AMPK and maintaining cellular, tissue and whole-body energy homeostasis