Circulating cell-free DNA in health and disease - the relationship to health behaviours, ageing phenotypes and metabolomics.

Circulating cell-free DNA (cf-DNA) has emerged as a promising biomarker of ageing, tissue damage and cellular stress. However, less is known about health behaviours, ageing phenotypes and metabolic processes that lead to elevated cf-DNA levels. We sought to analyse the relationship of circulating cf-DNA level to age, sex, smoking, physical activity, vegetable consumption, ageing phenotypes (physical functioning, the number of diseases, frailty) and an extensive panel of biomarkers including blood and urine metabolites and inflammatory markers in three human cohorts (N = 5385; 17-82 years). The relationships were assessed using correlation statistics, and linear and penalised regressions (the Lasso), also stratified by sex.cf-DNA levels were significantly higher in men than in women, and especially in middle-aged men and women who smoke, and in older more frail individuals. Correlation statistics of biomarker data showed that cf-DNA level was higher with elevated inflammation (C-reactive protein, interleukin-6), and higher levels of homocysteine, and proportion of red blood cells and lower levels of ascorbic acid. Inflammation (C-reactive protein, glycoprotein acetylation), amino acids (isoleucine, leucine, tyrosine), and ketogenesis (3-hydroxybutyrate) were included in the cf-DNA level-related biomarker profiles in at least two of the cohorts.In conclusion, circulating cf-DNA level is different by sex, and related to health behaviour, health decline and metabolic processes common in health and disease. These results can inform future studies where epidemiological and biological pathways of cf-DNA are to be analysed in details, and for studies evaluating cf-DNA as a potential clinical marker.

Senescent human melanocytes drive skin ageing via paracrine telomere dysfunction.

Cellular senescence has been shown to contribute to skin ageing. However, the role of melanocytes in the process is understudied. Our data show that melanocytes are the only epidermal cell type to express the senescence marker p16INK4A during human skin ageing. Aged melanocytes also display additional markers of senescence such as reduced HMGB1 and dysfunctional telomeres, without detectable telomere shortening. Additionally, senescent melanocyte SASP induces telomere dysfunction in paracrine manner and limits proliferation of surrounding cells via activation of CXCR3-dependent mitochondrial ROS. Finally, senescent melanocytes impair basal keratinocyte proliferation and contribute to epidermal atrophy in vitro using 3D human epidermal equivalents. Crucially, clearance of senescent melanocytes using the senolytic drug ABT737 or treatment with mitochondria-targeted antioxidant MitoQ suppressed this effect. In conclusion, our study provides proof-of-concept evidence that senescent melanocytes affect keratinocyte function and act as drivers of human skin ageing.

Reallocating sitting time to standing or stepping through isotemporal analysis: associations with markers of chronic low-grade inflammation.

Although high levels of sitting time are adversely related to health, it is unclear whether moving from sitting to standing provides a sufficient stimulus to elicit benefits upon markers of chronic low-grade inflammation in a population at high risk of type 2 diabetes (T2DM). Three hundred and seventy two participants (age = 66.8 ± 7.5years; body mass index (BMI) = 31.7 ± 5.5kg/m2; Male = 61%) were included. Sitting, standing and stepping was determined using the activPAL3TM device. Linear regression modelling employing an isotemporal substitution approach was used to quantify the association of theoretically substituting 60 minutes of sitting per day for standing or stepping on interleukin-6 (IL-6), C-reactive protein (CRP) and leptin. Reallocating 60 minutes of sitting time per day for standing was associated with a -4% (95% CI -7%, -1%) reduction in IL-6 (p = 0.048). Reallocating 60 minutes of sitting time for light stepping was also associated with lower IL-6 levels (-28% (-46%, -4%; p = 0.025)). Substituting sitting for moderate-to-vigorous (MVPA) stepping was associated with lower CRP (-41% (-75%, -8%; p = 0.032)), leptin (-24% (-34%, -12%; p ≤ 0.001)) and IL-6 (-16% (-28%, 10%; p = 0.036). Theoretically replacing 60 minutes of sitting per day with an equal amount of either standing or stepping yields beneficial associations upon markers of chronic-low grade inflammation.

Sedentary time and markers of chronic low-grade inflammation in a high risk population.

BACKGROUND: Sedentary behaviour has been identified as a distinct risk factor for several health outcomes. Nevertheless, little research has been conducted into the underlying mechanisms driving these observations. This study aimed to investigate the association of objectively measured sedentary time and breaks in sedentary time with markers of chronic low-grade inflammation and adiposity in a population at a high risk of type 2 diabetes mellitus. METHODS: This study reports data from an ongoing diabetes prevention programme conducted in Leicestershire, UK. High risk individuals were recruited from 10 primary care practices. Sedentary time (<25 counts per 15 s) was measured using Actigraph GT3X accelerometers (15 s epochs). A break was considered as any interruption in sedentary time (≥25 counts per 15 s). Biochemical outcomes included interleukin-6 (IL-6), C-reactive protein (CRP), leptin, adiponectin and leptin:adiponectin ratio (LAR). A sensitivity analysis investigated whether results were affected by removing participants with a CRP level >10 mg/L, as this can be indicative of acute inflammation. RESULTS: 558 participants (age = 63.6±7.7 years; male = 64.7%) had complete adipokine and accelerometer data. Following adjustment for various confounders, sedentary time was detrimentally associated with CRP (ß = 0.176±0.057, p = 0.002), IL-6 (ß = 0.242±0.056, p = <0.001), leptin (ß = 0.146±0.043, p = <0.001) and LAR (ß = 0.208±0.052, p = <0.001). Associations were attenuated after further adjustment for moderate-to-vigorous physical activity (MVPA) with only IL-6 (ß = 0.231±0.073, p = 0.002) remaining significant; this result was unaffected after further adjustment for body mass index and glycosylated haemoglobin (HbA1c). Similarly, breaks in sedentary time were significantly inversely associated with IL-6 (ß = -0.094±0.047, p = 0.045) and leptin (ß = -0.075±0.037, p = 0.039); however, these associations were attenuated after adjustment for accelerometer derived variables. Excluding individuals with a CRP level >10 mg/L consistently attenuated the significant associations across all markers of inflammation. CONCLUSION: These novel findings from a high risk population recruited through primary care suggest that sedentary behaviour may influence markers associated with inflammation, independent of MVPA, glycaemia and adiposity.

Effect of short-term reduced physical activity on cardiovascular risk factors in active lean and overweight middle-aged men.

OBJECTIVES: An experimental reduction in physical activity is a useful tool for exploring the health benefits of physical activity. This study investigated whether similarly-active overweight men show a more pronounced response to reduced physical activity than their lean counterparts because of their atherogenic phenotype (i.e., greater abdominal adiposity). METHODS: From 115 active men aged 45-64years, we recruited nine active lean (waist circumference <84cm) and nine active central overweight men (waist circumference >94cm). Fasting blood samples and responses to an oral glucose tolerance test (OGTT) were measured at baseline and following one week of reduced physical activity to simulate sedentary levels (removal of structured exercise and reduced habitual physical activity). RESULTS: Glucose and insulin areas under the curve (AUC), CRP, ALT, TAG were all higher in the overweight group and remained so throughout (P<0.05). Insulin and glucose AUC responses to an OGTT, as well as fasting triglyceride (TAG) concentrations, increased in both groups as a result of the intervention (P<0.05). There was no change in interleukin-6, C-reactive protein (CRP), Tumour Necrosis Factor-α, soluble intracellular adhesion molecule 1, or alanine transaminase (ALT). CONCLUSION: One-week of reduced activity similarly-impaired glucose control and increased fasting TAG in both lean and overweight men. Importantly, in spite of very similar (high) levels of habitual physical activity, central overweight men displayed a poorer profile for various inflammatory and metabolic outcomes (CRP, ALT, TAG, glucose AUC and insulin AUC).

Self-reported sitting time and markers of inflammation, insulin resistance, and adiposity.

BACKGROUND: Sedentary behavior is emerging as an independent risk factor for chronic disease; however, potential mechanisms underpinning these observations are not well understood. PURPOSE: This study aimed to investigate the association of self-reported weekday sitting time with biomarkers linked to chronic low-grade inflammation, insulin resistance, and adiposity. METHODS: This study reports data from individuals attending a diabetes screening program, United Kingdom, 2004-2007; analysis was conducted in 2010. Sitting time and physical activity were measured using the International Physical Activity Questionnaire; biochemical outcomes included fasting and 2-hour postchallenge glucose, fasting insulin, C-reactive protein (CRP), leptin, adiponectin, and interleukin-6 (IL-6). RESULTS: This study included 505 (female=46%; South-Asian ethnicity=19%, aged 59±10 years, BMI=29.5±4.7) individuals with valid sitting data. Increased sitting time was positively associated with fasting insulin, leptin, leptin/adiponectin ratio, CRP, and IL-6 in women, but not men, after adjustment for age, ethnicity, social deprivation, and smoking and medication status; interaction analysis revealed that the gender-specific differences were significant. The associations for women remained significant after additional adjustment for total moderate- to vigorous-intensity physical activity; however all associations were attenuated when further adjusted for BMI. There was no association between sitting time and glycemic status. CONCLUSIONS: Total self-reported weekday sitting time was associated with biomarkers linked to chronic low-grade inflammation and poor metabolic health in women, but not men, independent of physical activity.

Active middle-aged men have lower fasting inflammatory markers but the postprandial inflammatory response is minimal and unaffected by physical activity status.

Physical activity modifies some postprandial responses such as glycemic control, although it is unclear whether this translates into lower postprandial inflammation. Our objective in this study was to determine whether postprandial inflammatory markers are lower in active compared with sedentary middle-aged men. Thirteen active and twelve sedentary middle-aged men consumed a mixed meal on one occasion. Blood was taken via a cannula before and up to 8 h after the meal and with a single-use needle before and 8 h after the meal. Active men had lower fasted IL-6 (0.6 +/- 0.2 vs. 1.2 +/- 0.3 pg/ml; P = 0.004) and C-reactive protein (1.3 +/- 0.3 vs. 2.9 +/- 0.6 mg/l; P = 0.04) concentrations than sedentary men. Cannula blood IL-6 concentrations increased by 3.49 pg/ml in the 8 h following the meal (P < 0.001); however, this increase was minimal (0.36 pg/ml) in blood taken via a single-use needle from the contralateral arm (P = 0.013). The sedentary group had larger glucose (P = 0.034), insulin (P = 0.013), and triacylglycerol (P = 0.057) responses to the meal. These results provide further evidence that physical activity is associated with lower inflammatory marker concentrations in a fasted state and a lower postprandial metabolic response to a meal. However, this does not translate into lower postprandial inflammatory markers since the only evidence of postprandial inflammation (a large increase in serum IL-6) was actually due to the cannula used for blood sampling.

Acute consumption of fish oil improves postprandial VLDL profiles in healthy men aged 50-65 years.

Dietary supplementation with fish oil induces beneficial changes in the size and concentration of plasma lipoproteins, although the underlying mechanism is unclear. We have investigated the effect of increasing the amount of fish oil in a single meal on the size and concentration of VLDL, LDL and HDL particles during the postprandial period. Healthy men aged 58 (sd 5) years (n 11) consumed isoenergetic, mixed macronutrient test meals containing either 0.3 g (reference, REF) or 2.2 g (high fish oil, HFO) fish oil in a randomised order, and blood samples were collected over the following 6 h. Plasma lipoprotein size and concentration were measured by NMR spectroscopy. There was a significant interaction effect of time and meal composition on the VLDL, but not on the LDL or HDL, concentration (P = 0.036) and particle size (P = 0.005). Consuming the HFO meal significantly increased the VLDL concentration (P < 0.05) and reduced VLDL particle size (P < 0.05) when compared with the REF meal and baseline. LDL particle size decreased slightly during the postprandial period, but there was no difference between the meals. There was no effect of time or meal composition in the LDL concentration. The HDL concentration decreased and size increased slightly during the postprandial period, but there were no significant differences between the meals. Increased consumption of fish oil induces acute changes in the VLDL, but not in the LDL or HDL, metabolism.

Walking and inflammatory markers in individuals screened for type 2 diabetes.

OBJECTIVE: To investigate the association of walking activity with inflammatory markers and fasting insulin in a bi-ethnic population screened for type 2 diabetes in Leicester, United Kingdom, between 2005 and 2006. METHOD: Physical activity, adipocytokine, high-sensitivity C-reactive protein and fasting insulin measurements were available for 400 individuals screened for type 2 diabetes. Of the 400 participants, 56% were diagnosed with normal glucose control, 36% with prediabetes and 8% with diabetes. RESULTS: Multivariate statistical analysis showed that those who reported walking for at least 30 min on at least 5 days/week had lower levels of C-reactive protein, interleukin-6, and tumor necrosis factor-alpha compared to those who reported lower walking activity levels, after adjustment for other modes of moderate-to-vigorous intensity physical activity, age, ethnicity, sex, social deprivation and smoking status. Further adjustment for waist circumference attenuated the association of walking with tumor necrosis factor-alpha. CONCLUSION: Walking activity, independent of other forms of physical activity, is associated with lower levels of circulating pro-inflammatory markers.

Soy isoflavones increase preprandial peptide YY (PYY), but have no effect on ghrelin and body weight in healthy postmenopausal women.

BACKGROUND: Soy isoflavones show structural and functional similarities to estradiol. Available data indicate that estradiol and estradiol-like components may interact with gut "satiety hormones" such as peptide YY (PYY) and ghrelin, and thus influence body weight. In a randomized, double-blind, placebo-controlled, cross-over trial with 34 healthy postmenopausal women (59 +/- 6 years, BMI: 24.7 +/- 2.8 kg/m2), isoflavone-enriched cereal bars (50 mg isoflavones/day; genistein to daidzein ratio 2:1) or non-isoflavone-enriched control bars were consumed for 8 weeks (wash-out period: 8-weeks). Seventeen of the subjects were classified as equol producers. Plasma concentrations of ghrelin and PYY, as well as energy intake and body weight were measured at baseline and after four and eight weeks of each intervention arm. RESULTS: Body weight increased in both treatment periods (isoflavone: 0.40 +/- 0.94 kg, P < 0.001; placebo: 0.66 +/- 0.87 kg, P = 0.018), with no significant difference between treatments. No significant differences in energy intake were observed (P = 0.634). PYY significantly increased during isoflavone treatment (51 +/- 2 pmol/L vs. 55 +/- 2 pmol/L), but not during placebo (52 +/- 3 pmol/L vs. 50 +/- 2 pmol/L), (P = 0.010 for treatment differences, independent of equol production). Baseline plasma ghrelin was significantly lower in equol producers (110 +/- 16 pmol/L) than in equol non-producers (162 +/- 17 pmol/L; P = 0.025). CONCLUSION: Soy isoflavone supplementation for eight weeks did not significantly reduce energy intake or body weight, even though plasma PYY increased during isoflavone treatment. Ghrelin remained unaffected by isoflavone treatment. A larger and more rigorous appetite experiment might detect smaller differences in energy intake after isoflavone consumption. However, the results of the present study do not indicate that increased PYY has a major role in the regulation of body weight, at least in healthy postmenopausal women.

Soy-isoflavone-enriched foods and markers of lipid and glucose metabolism in postmenopausal women: interactions with genotype and equol production.

BACKGROUND: The hypocholesterolemic effects of soy foods are well established, and it has been suggested that isoflavones are responsible for this effect. However, beneficial effects of isolated isoflavones on lipid biomarkers of cardiovascular disease risk have not yet been shown. OBJECTIVE: The objective was to investigate the effects of isolated soy isoflavones on metabolic biomarkers of cardiovascular disease risk, including plasma total, HDL, and LDL cholesterol; triacylglycerols; lipoprotein(a); the percentage of small dense LDL; glucose; nonesterified fatty acids; insulin; and the homeostasis model assessment of insulin resistance. Differences with respect to single nucleotide polymorphisms in selected genes [ie, estrogen receptor alpha (XbaI and PvuII), estrogen receptor beta (AluI), and estrogen receptor beta(cx) (Tsp509I), endothelial nitric oxide synthase (Glu298Asp), apolipoprotein E (Apo E2, E3, and E4), cholesteryl ester transfer protein (TaqIB), and leptin receptor (Gln223Arg)] and with respect to equol production were investigated. DESIGN: Healthy postmenopausal women (n = 117) participated in a randomized, double-blind, placebo-controlled, crossover dietary intervention trial. Isoflavone-enriched (genistein-to-daidzein ratio of 2:1; 50 mg/d) or placebo cereal bars were consumed for 8 wk, with a wash-out period of 8 wk before the crossover. RESULTS: Isoflavones did not have a significant beneficial effect on plasma concentrations of lipids, glucose, or insulin. A significant difference between the responses of HDL cholesterol to isoflavones and to placebo was found with estrogen receptor beta(cx) Tsp509I genotype AA, but not GG or GA. CONCLUSIONS: Isoflavone supplementation, when provided in the form and dose used in this study, had no effect on lipid or other metabolic biomarkers of cardiovascular disease risk in postmenopausal women but may increase HDL cholesterol in an estrogen receptor beta gene-polymorphic subgroup.

Soy-isoflavone-enriched foods and inflammatory biomarkers of cardiovascular disease risk in postmenopausal women: interactions with genotype and equol production.

BACKGROUND: Dietary isoflavones are thought to be cardioprotective because of their structural similarity to estrogen. The reduction of concentrations of circulating inflammatory markers by estrogen may be one of the mechanisms by which premenopausal women are protected against cardiovascular disease. OBJECTIVE: Our aim was to investigate the effects of isolated soy isoflavones on inflammatory biomarkers [von Willebrand factor, intracellular adhesion molecule 1, vascular cell adhesion molecule 1 (VCAM-1), E-selectin, monocyte chemoattractant protein 1, C-reactive protein (CRP), and endothelin 1 concentrations]. Differences with respect to single-nucleotide polymorphisms in selected genes [estrogen receptor alpha (XbaI and PvuII), estrogen receptor beta [ERbeta (AluI) and ERbeta[cx] (Tsp509I), endothelial nitric oxide synthase (Glu298Asp), apolipoprotein E (Apo E2, E3, and E4), and cholesteryl ester transfer protein (TaqIB)] and equol production were investigated. DESIGN: One hundred seventeen healthy European postmenopausal women participated in this randomized, double-blind, placebo-controlled, crossover dietary intervention trial. Isoflavone-enriched (genistein-to-daidzein ratio of 2:1; 50 mg/d) or placebo cereal bars were consumed for 8 wk, with a washout period of 8 wk between the crossover. Plasma inflammatory factors were measured at 0 and 8 wk of each study arm. RESULTS: Isoflavones improved CRP concentrations [odds ratio (95% CI) for CRP values >1 mg/L for isoflavone compared with placebo: 0.43 (0.27, 0.69)]; no significant effects of isoflavone treatment on other plasma inflammatory markers were observed. No significant differences in the response to isoflavones were observed according to subgroups of equol production. Differences in the VCAM-1 response to isoflavones and to placebo were found with ERbeta AluI genotypes. CONCLUSION: Isoflavones have beneficial effects on CRP concentrations, but not on other inflammatory biomarkers of cardiovascular disease risk in postmenopausal women, and may improve VCAM-1 in an ERbeta gene polymorphic subgroup.