DNA helicases Sgs1 and BLM promote DNA double-strand break resection

A key cellular response to DNA double-strand breaks (DSBs) is 5'-to-3' DSB resection by nucleases to generate regions of ssDNA that then trigger cell cycle checkpoint signaling and DSB repair by homologous recombination (HR). Here, we reveal that in the absence of exonuclease Exo1 activity, deletion or mutation of the Saccharomyces cerevisiae RecQ-family helicase, Sgs1, causes pronounced hypersensitivity to DSB-inducing agents. Moreover, we establish that this reflects severely compromised DSB resection, deficient DNA damage signaling, and strongly impaired HR-mediated repair. Furthermore, we show that the mammalian Sgs1 ortholog, BLM--whose deficiency causes cancer predisposition and infertility in people--also functions in parallel with Exo1 to promote DSB resection, DSB signaling and resistance to DSB-generating agents. Collectively, these data establish evolutionarily conserved roles for the BLM and Sgs1 helicases in DSB processing, signaling, and repair.     

Perivascular Drainage of Amyloid-β Peptides from the Brain and Its Failure in Cerebral Amyloid Angiopathy and Alzheimer's Disease

Alzheimer's disease is the commonest dementia. One major characteristic of its pathology is accumulation of amyloid-β (Aβ) as insoluble deposits in brain parenchyma and in blood vessel walls [cerebral amyloid angiopathy (CAA)]. The distribution of Aβ deposits in the basement membranes of cerebral capillaries and arteries corresponds to the perivascular drainage pathways by which interstitial fluid (ISF) and solutes are eliminated from the brain—effectively the lymphatic drainage of the brain. Theoretical models suggest that vessel pulsations supply the motive force for perivascular drainage of ISF and solutes. As arteries stiffen with age, the amplitude of pulsations is reduced and insoluble Aβ is deposited in ISF drainage pathways as CAA, thus, further impeding the drainage of soluble Aβ. Failure of perivascular drainage of Aβ and deposition of Aβ in the walls of arteries has two major consequences: (i) intracerebral hemorrhage associated with rupture of Aβ-laden arteries in CAA; and (ii) Alzheimer's disease in which failure of elimination of ISF, Aβ and other soluble metabolites from the brain alters homeostasis and the neuronal environment resulting in cognitive decline and dementia. Therapeutic strategies that improve elimination of Aβ and other soluble metabolites from the brain may prevent cognitive decline in Alzheimer's disease.     

AMP-activated protein kinase signalling pathways are down regulated and skeletal muscle development impaired in fetuses of obese, over-nourished sheep

Maternal obesity and over-nutrition give rise to both obstetric problems and neonatal morbidity. The objective of this study was to evaluate effects of maternal obesity and over-nutrition on signalling of the AMP-activated protein kinase (AMPK) pathway in fetal skeletal muscle in an obese pregnant sheep model. Non-pregnant ewes were assigned to a control group (Con, fed 100% of NRC nutrient recommendations, n = 7) or obesogenic group (OB, fed 150% of National Research Council (NRC) recommendations, n = 7) diet from 60 days before to 75 days after conception (term 150 days) when fetal semitendinosus skeletal muscle (St) was sampled. OB mothers developed severe obesity accompanied by higher maternal and fetal plasma glucose and insulin levels. In fetal St, activity of phosphoinositide-3 kinase (PI3K) associated with insulin receptor substrate-1 (IRS-1) was attenuated ( P < 0.05), in agreement with the increased phophorylation of IRS-1 at serine 1011. Phosphorylation of AMP-activated protein kinase (AMPK) at Thr 172, acetyl-CoA carboxylase at Ser 79, tuberous sclerosis 2 at Thr 1462 and eukaryotic translation initiation factor 4E-binding protein 1 at Thr 37/46 were reduced in OB compared to Con fetal St. No difference in energy status (AMP/ATP ratio) was observed. The expression of protein phosphatase 2C was increased in OB compared to Con fetal St. Plasma tumour necrosis factor α (TNFα) was increased in OB fetuses indicating an increased inflammatory state. Expression of peroxisome proliferator-activated receptor γ (PPARγ) was higher in OB St, indicating enhanced adipogenesis. The glutathione: glutathione disulphide ratio was also lower, showing increased oxidative stress in OB fetal St. In summary, we have demonstrated decreased signalling of the AMPK system in skeletal muscle of fetuses of OB mothers, which may play a role in altered muscle development and development of insulin resistance in the offspring.     

Vascular smooth muscle cells undergo telomere-based senescence in human atherosclerosis: effects of telomerase and oxidative stress

Although human atherosclerosis is associated with aging, direct evidence of cellular senescence and the mechanism of senescence in vascular smooth muscle cells (VSMCs) in atherosclerotic plaques is lacking. We examined normal vessels and plaques by histochemistry, Southern blotting, and fluorescence in situ hybridization for telomere signals. VSMCs in fibrous caps expressed markers of senescence (senescence-associated beta-galactosidase [SAbetaG] and the cyclin-dependent kinase inhibitors [cdkis] p16 and p21) not seen in normal vessels. In matched samples from the same individual, plaques demonstrated markedly shorter telomeres than normal vessels. Fibrous cap VSMCs exhibited markedly shorter telomeres compared with normal medial VSMCs. Telomere shortening was closely associated with increasing severity of atherosclerosis. In vitro, plaque VSMCs demonstrated morphological features of senescence, increased SAbetaG expression, reduced proliferation, and premature senescence. VSMC senescence was mediated by changes in cyclins D/E, p16, p21, and pRB, and plaque VSMCs could reenter the cell cycle by hyperphosphorylating pRB. Both plaque and normal VSMCs expressed low levels of telomerase. However, telomerase expression alone rescued plaque VSMC senescence despite short telomeres, normalizing the cdki/pRB changes. In vivo, plaque VSMCs exhibited oxidative DNA damage, suggesting that telomere damage may be induced by oxidant stress. Furthermore, oxidants induced premature senescence in vitro, with accelerated telomere shortening and reduced telomerase activity. We conclude that human atherosclerosis is characterized by senescence of VSMCs, accelerated by oxidative stress-induced DNA damage, inhibition of telomerase and marked telomere shortening. Prevention of cellular senescence may be a novel therapeutic target in atherosclerosis.     

PPARgamma and C/EBP factors orchestrate adipocyte biology via adjacent binding on a genome-wide scale

Peroxisome proliferator-activated receptor γ(PPARγ), a nuclear receptor and the target of anti-diabetic thiazolinedione drugs, is known as the master regulator of adipocyte biology. Although it regulates hundreds of adipocyte genes, PPARγ binding to endogenous genes has rarely been demonstrated. Here, utilizing chromatin immunoprecipitation (ChIP) coupled with whole genome tiling arrays, we identified 5299 genomic regions of PPARγ binding in mouse 3T3-L1 adipocytes. The consensus PPARγ/RXRα “DR-1”-binding motif was found at most of the sites, and ChIP for RXRα showed colocalization at nearly all locations tested. Bioinformatics analysis also revealed CCAAT/enhancer-binding protein (C/EBP)-binding motifs in the vicinity of most PPARγ-binding sites, and genome-wide analysis of C/EBPα binding demonstrated that it localized to 3350 of the locations bound by PPARγ. Importantly, most genes induced in adipogenesis were bound by both PPARγ and C/EBPα, while very few were PPARγ-specific. C/EBPβ also plays a role at many of these genes, such that both C/EBPα and β are required along with PPARγ for robust adipocyte-specific gene expression. Thus, PPARγ and C/EBP factors cooperatively orchestrate adipocyte biology by adjacent binding on an unanticipated scale.     

Microstimulation of monkey dorsolateral prefrontal cortex impairs antisaccade performance

The dorsolateral prefrontal cortex (DLPFC) has been implicated in various cognitive functions, including response suppression. This function is frequently probed with the antisaccade task, which requires suppression of the automatic tendency to look toward a flashed peripheral stimulus (prosaccade), and instead generate a voluntary saccade to the mirror location. To test whether activity in the DLPFC is causally linked to antisaccade performance, we applied electrical microstimulation to sites in the DLPFC of two monkeys, while they performed randomly interleaved pro- and antisaccade trials. Microstimulation resulted in significantly longer saccadic reaction times for ipsilaterally directed prosaccades and antisaccades, and increased the error rate on ipsilateral antisaccade trials. These findings provide causal evidence that activity in the DLPFC influences saccadic eye movements.     

Differential effects of n-3 polyunsaturated fatty acids on metabolic control and vascular reactivity in the type 2 diabetic ob/ob mouse

Diets rich in monounsaturated fatty acids (MUFA) are recommended for individuals with type 2 diabetes mellitus (T2DM). The American Heart Association recommends increasing intakes of n-3 polyunsaturated fatty acids (PUFA) to reduce the risk of vascular disease in high-risk individuals; however, the long-term effects of these bioactive fatty acids on glucose metabolism in insulin resistance are controversial. The present studies were conducted to evaluate the effects of diets rich in both MUFA and alpha linolenic acid (C18:3n-3, ALA), eicosapentaenoic acid (C20:5n-3, EPA), or docosahexaenoic acid (C22:6n-3, DHA), on glycemic control and other parameters related to vascular health in a mouse model of T2DM and insulin resistance. Male ob/ob mice (n = 15 per treatment) were fed 1 of 4 lipid-modified formula diets (LFDs) for 4 weeks: (1) MUFA control, (2) ALA blend, (3) EPA blend, and (4) DHA blend. A portion of a MUFA-rich lipid blend in the control LFD was replaced with 11% to 14% energy as n-3 PUFA. After 4 weeks, plasma glucose response to a standard meal (1.5 g carbohydrate/kg body weight) and insulin challenge (2 U/kg body weight, IP) was assessed, and samples were collected for analysis of glucose, insulin, and lipids. Vascular reactivity of isolated aortic rings was assessed in an identical follow-up study. The results showed that insulin-resistant mice fed an LFD with EPA and/or DHA blends had significantly (P < .05) lower triglycerides and free fatty acids, but insulin sensitivity and fasting plasma glucose were not improved. However, mice fed with the ALA blend had significantly improved insulin sensitivity when compared to those fed with other LFD (P < .05). Animals fed an LFD with n-3 PUFA from marine or plant sources showed significantly improved vascular responses as compared with the MUFA-rich LFD (E(max), P < .05) and ob/ob reference mice consuming chow (E(max) and pEC(50), P < .05). In summary, long-term consumption of LFD with n-3 PUFAs improved blood lipids and vascular function in an animal model of insulin resistance and T2DM; however, only MUFA-rich LFD with ALA also improved both insulin sensitivity and glycemic responses. Further studies of MUFA-rich LFD with ALA with individuals who have T2DM are warranted.     

Increasing objectively measured sedentary time increases clustered cardiometabolic risk: a 6 year analysis of the ProActive study

Aims/hypothesis

We aimed to quantify the associations between change in objectively measured sedentary and moderate-to-vigorous physical activity (MVPA) times and self-reported television viewing over 6 years and change in a clustered cardiometabolic risk score (CCMR), including and excluding waist circumference (CCMR without adiposity component, CCMR _{no adip} ), and its individual components, among the adult children of people with type 2 diabetes.

Methods

In 171 adults (mean ± SD age 42.52?±?6.30 years; 46% men) with a parental history of diabetes (ProActive UK), physical activity accelerometer measures and self-reported television viewing were assessed at baseline and a mean ± SD of 6.27?±?0.46 years later. Associations between change in sedentary time, MVPA time and television viewing and cardiometabolic risk and mediation by adiposity change were examined by multiple linear regression and the product of coefficients method, respectively.

Results

Greater increases in sedentary time (h/day) were associated with larger increases in clustered cardiometabolic risk (CCMR: 0.08 [95% CI 0.01, 0.15]; CCMR _{no adip} : 0.08 [0.01, 0.16]) and triacylglycerol (0.15 [0.01, 0.29]), independent of baseline sedentary and MVPA times, change in MVPA time and other confounders. No evidence was found for mediation by change in waist circumference and BMI for the associations with CCMR _{no adip} and triacylglycerol. Greater increases in MVPA time (h/day) were associated with larger decreases in waist circumference (?3.86 [?7.58, ?0.14]), independently of baseline MVPA and sedentary times, change in sedentary time and other confounders. Television viewing was not independently associated with any of the cardiometabolic outcomes.

Conclusions/interpretation

Increasing sedentary time is independently related to increasing clustered cardiometabolic risk and triacylglycerol in adults at high risk of developing diabetes. Strategies to prevent diabetes might target reducing sedentary time. Trial registration ISRCTN61323766