Proton magnetic resonance spectroscopy shows lower intramyocellular lipid accumulation in middle-aged subjects predisposed to familial longevity

Families predisposed to longevity show enhanced glucose tolerance and skeletal muscle insulin sensitivity compared with controls, independent of body composition and physical activity. Intramyocellular lipid (IMCL) accumulation in skeletal muscle has been associated with insulin resistance. Here, we assessed whether subjects enriched for familial longevity have lower IMCL levels. We determined IMCL levels in 48 subjects from the Leiden Longevity Study, comprising 24 offspring of nonagenarian siblings and 24 partners thereof as control subjects. IMCL levels were assessed noninvasively using short echo time proton magnetic resonance spectroscopy ((1)H-MRS) of the tibialis anterior muscle with a 7 Tesla human MR scanner. IMCL levels were calculated relative to the total creatine (tCr) CH3 signal. Physical activity was assessed using the International Physical Activity Questionnaire (IPAQ). After correction for age, sex, BMI, and physical activity, offspring of long-lived nonagenarian siblings tended to show lower IMCL levels compared with controls (IMCL/tCr: 3.1 ± 0.5 vs. 4.5 ± 0.5, respectively, P = 0.051). In a pairwise comparison, this difference reached statistical significance (P = 0.038). We conclude that offspring of nonagenarian siblings predisposed to longevity show lower IMCL levels compared with environmentally matched control subjects. Future research should focus on assessing what mechanisms may explain the lower IMCL levels in familial longevity.     

Transcriptional profiling of human familial longevity indicates a role for ASF1A and IL7R

The Leiden Longevity Study consists of families that express extended survival across generations, decreased morbidity in middle-age, and beneficial metabolic profiles. To identify which pathways drive this complex phenotype of familial longevity and healthy aging, we performed a genome-wide gene expression study within this cohort to screen for mRNAs whose expression changes with age and associates with longevity. We first compared gene expression profiles from whole blood samples between 50 nonagenarians and 50 middle-aged controls, resulting in identification of 2,953 probes that associated with age. Next, we determined which of these probes associated with longevity by comparing the offspring of the nonagenarians (50 subjects) and the middle-aged controls. The expression of 360 probes was found to change differentially with age in members of the long-lived families. In a RT-qPCR replication experiment utilizing 312 controls, 332 offspring and 79 nonagenarians, we confirmed a nonagenarian specific expression profile for 21 genes out of 25 tested. Since only some of the offspring will have inherited the beneficial longevity profile from their long-lived parents, the contrast between offspring and controls is expected to be weak. Despite this dilution of the longevity effects, reduced expression levels of two genes, ASF1A and IL7R, involved in maintenance of chromatin structure and the immune system, associated with familial longevity already in middle-age. The size of this association increased when controls were compared to a subfraction of the offspring that had the highest probability to age healthily and become long-lived according to beneficial metabolic parameters. In conclusion, an "aging-signature" formed of 21 genes was identified, of which reduced expression of ASF1A and IL7R marked familial longevity already in middle-age. This indicates that expression changes of genes involved in metabolism, epigenetic control and immune function occur as a function of age, and some of these, like ASF1A and IL7R, represent early features of familial longevity and healthy ageing.     

Human in vivo longevity is reflected in vitro by differential metabolism as measured by (1)H-NMR profiling of cell culture supernatants

The offspring of nonagenarian siblings suffer less from age related conditions and have a lower risk of mortality compared to their partners. Fibroblast strains derived from such offspring in middle age show different in vitro responses to stress, more stress-induced apoptosis and less senescence when compared to strains of their partners. Aiming to find differences in cellular metabolism in vitro between these fibroblast strains, cell culture supernatants collected at 24 hours and five days were analysed using (1)H nuclear magnetic resonance (NMR)-based metabolic footprinting. Between 24 hours and five days of incubation, supernatants of all fibroblast strains showed decreased levels of glucose, pyruvate, alanine-glutamine (ala-gln), valine, leucine, isoleucine, serine and lysine and increased levels of glutamine, alanine, lactate and pyroglutamic acid. Strains from offspring and their partners were compared using a partial least squares-discriminant analysis (PLS-DA) model based on the data of the five-day time point. The ala-gln and glucose consumption were higher for fibroblast strains derived from offspring when compared to strains of their partners. Also, production of glutamine, alanine, lactate and pyroglutamic acid was found to be higher for fibroblast strains derived from offspring. In conclusion, differences in NMR-based metabolic profiles of human cells in vitro reflect the propensity for human longevity of the subjects from whom these were derived.     

Plasma protein N-glycan profiles are associated with calendar age, familial longevity and health

The development of medical interventions for the preservation of disease-free longevity would be facilitated by markers that predict healthy aging. Altered protein N-glycosylation patterns have been found with increasing age and several disease states. Here we investigate whether glycans derived from the total glycoprotein pool in plasma mark familial longevity and distinguish healthy from unhealthy aging. Total plasma N-glycan profiles of 2396 middle aged participants in the Leiden Longevity Study (LLS) were obtained by glycan release, labeling, and subsequent HPLC analysis with fluorescence detection. After normalization and batch correction, several regression strategies were applied to evaluate associations between glycan patterns, familial longevity, and healthy aging. Two N-glycan features (LC-7 and LC-8) were identified to be more abundant in plasma of the offspring of long-lived individuals as compared to controls. These results were not confounded by the altered lipid status or glucose homeostasis of the offspring. Furthermore, a decrease in levels of LC-8 was associated with the occurrence of myocardial infarction (p = 0.049, coefficient = -0.065), indicating that plasma glycosylation patterns do not only mark familial longevity but may also reflect healthy aging. In conclusion, we describe two glycan features, of which increased levels mark familial longevity and decreased levels of one of these features mark the presence of cardiovascular disease.     

Growth hormone secretion is diminished and tightly controlled in humans enriched for familial longevity

Reduced growth hormone (GH) signaling has been consistently associated with increased health and lifespan in various mouse models. Here, we assessed GH secretion and its control in relation with human familial longevity. We frequently sampled blood over 24 h in 19 middle‐aged offspring of long‐living families from the Leiden Longevity Study together with 18 of their partners as controls. Circulating GH concentrations were measured every 10 min and insulin‐like growth factor 1 (IGF‐1) and insulin‐like growth factor binding protein 3 (IGFBP3) every 4 h. Using deconvolution analysis, we found that 24‐h total GH secretion was 28% lower (P = 0.04) in offspring [172 (128–216) mU L^?1] compared with controls [238 (193–284) mU L^?1]. We used approximate entropy (ApEn) to quantify the strength of feedback/feedforward control of GH secretion. ApEn was lower (P = 0.001) in offspring [0.45 (0.39–0.53)] compared with controls [0.66 (0.56–0.77)], indicating tighter control of GH secretion. No significant differences were observed in circulating levels of IGF‐1 and IGFBP3 between offspring and controls. In conclusion, GH secretion in human familial longevity is characterized by diminished secretion rate and more tight control. These data imply that the highly conserved GH signaling pathway, which has been linked to longevity in animal models, is also associated with human longevity.     

Brain tissue volumes in familial longevity: the Leiden Longevity Study

Atrophy is one of the major age‐related changes in the brain. The absence of brain atrophy in elderly individuals reflects deceleration in the process of biological aging. Moreover, results from human twin studies suggest a large genetic influence on the variance of human brain tissue volumes. To investigate the association of brain volumes with exceptional longevity, we tested whether middle‐aged to elderly offspring of nonagenarian siblings have larger brain volumes than their spouses using magnetic resonance imaging. No differences in whole brain, gray matter and white matter volume were found. These brain volumes were associated with chronological age in offspring and control subjects (all P < 0.001). Left amygdalar volume of the offspring was larger (P = 0.03) compared with control subjects [mean volume offspring (cm³) (95% confidence interval, CI) = 1.39 (1.36–1.42), mean volume control subjects (cm³) (95% CI) = 1.32 (1.29–1.35)]. Association of left amygdalar volume with familial longevity was particularly pronounced when offspring with the oldest long‐lived parent were compared with control subjects (P = 0.01). Amygdalar volumes were not associated with chronological age in both groups. Our findings suggest that the observed association of a larger left amygdalar volume with familial longevity is not caused by a relative preservation of the left amygdala during the course of aging but most likely a result of early development caused by a genetic familial trait.     

Familial Longevity Is Marked by Better Cognitive Performance at Middle Age: The Leiden Longevity Study

Background

Decline in cognitive performance is a highly prevalent health condition in elderly. We studied whether offspring of nonagenarian siblings with a familial history of longevity, perform better on cognitive tests compared to their partners as controls. This is relevant since it could provide insights into determinants underlying decline in cognitive performance.

Methods

Cross-sectional analysis within the longitudinal cohort of the Leiden Longevity Study consisting of middle-aged offspring of nonagenarian siblings together with their partners (n = 500, mean age (SD) 66.3 (6.1) and 65.7 (7.2) years, respectively) as controls. Memory function, attention and processing speed were tested using the 15-Picture Learning Test, Stroop test and Digit Symbol Substitution Test. Data were analyzed with regression adjusted for age, gender, years of education and additionally for diabetes mellitus, cardiovascular diseases, alcohol use, smoking, inflammatory markers and apolipoprotein E genotype. Robust standard errors were used to account for familial relationships among the offspring.

Results

Cognitive performance was worse at higher calendar age (p<0.001, all except Stroop test part 1). The offspring performed better compared to their partners on trial 3 (p = 0.005), the immediate (p = 0.016) and delayed (p = 0.004) recall of the 15-Picture Learning Test as well as on the interference and combined interference score of the Stroop test (p = 0.014 and p = 0.036, respectively) in the fully adjusted model. The difference between offspring and partners was estimated to be more than three years according to the observed difference in calendar age.

Conclusions

Offspring of nonagenarian siblings with a familial history of longevity have better cognitive performance compared to the group of their partners of comparable age. This effect is independent of age-related diseases and known possible confounders. Possible explanations might be differences in subclinical vascular pathology between both groups.     

Lipidomics of familial longevity

Middle‐aged offspring of nonagenarians, as compared to their spouses (controls), show a favorable lipid metabolism marked by larger LDL particle size in men and lower total triglyceride levels in women. To investigate which specific lipids associate with familial longevity, we explore the plasma lipidome by measuring 128 lipid species using liquid chromatography coupled to mass spectrometry in 1526 offspring of nonagenarians (59 years ± 6.6) and 675 (59 years ± 7.4) controls from the Leiden Longevity Study. In men, no significant differences were observed between offspring and controls. In women, however, 19 lipid species associated with familial longevity. Female offspring showed higher levels of ether phosphocholine (PC) and sphingomyelin (SM) species (3.5–8.7%) and lower levels of phosphoethanolamine PE (38:6) and long‐chain triglycerides (TG) (9.4–12.4%). The association with familial longevity of two ether PC and four SM species was independent of total triglyceride levels. In addition, the longevity‐associated lipid profile was characterized by a higher ratio of monounsaturated (MUFA) over polyunsaturated (PUFA) lipid species, suggesting that female offspring have a plasma lipidome less prone to oxidative stress. Ether PC and SM species were identified as novel longevity markers in females, independent of total triglycerides levels. Several longevity‐associated lipids correlated with a lower risk of hypertension and diabetes in the Leiden Longevity Study cohort. This sex‐specific lipid signature marks familial longevity and may suggest a plasma lipidome with a better antioxidant capacity, lower lipid peroxidation and inflammatory precursors, and an efficient beta‐oxidation function.     

Nonsyndromic orofacial clefts: association with maternal hyperhomocysteinemia.

Maternal folic acid supplementation has been suggested to play a role in the prevention of nonsyndromic orofacial clefts, i.e., cleft lip +/- cleft palate. Using a case-control design, we investigated vitamin-dependent homocysteine metabolism in 35 mothers with nonsyndromic orofacial cleft offspring and 56 control mothers with nonmalformed offspring. A standardized oral methionine loading test was performed, in which fasting and afterload plasma total homocysteine, serum and red-cell folate, serum vitamin B12, and whole-blood vitamin B6 levels were determined. We found that both fasting (P < 0.01) as well as afterload (P < 0.05) homocysteine concentrations were significantly higher in cases compared to controls. Hyperhomocysteinemia, defined by a fasting and/or afterload homocysteine concentration above the 97.5th percentile, was present in 15.6% of the cases and in 3.6% of controls (odds ratio, 5.3 (1.1-24.2)). The median concentrations of serum (P < 0. 01) and red-cell (P < 0.05) folate were significantly higher, and vitamin B6 concentrations appeared to be significantly lower (P < 0. 05), in cases compared with controls. No significant difference was observed between groups for vitamin B12. These preliminary data offer evidence that maternal hyperhomocysteinemia may be a risk factor for having nonsyndromic orofacial cleft offspring.     

Familial longevity is characterized by high circadian rhythmicity of serum cholesterol in healthy elderly individuals

The biological clock, whose function deteriorates with increasing age, determines bodily circadian (i.e. 24h) rhythms, including that of cholesterol metabolism. Dampening of circadian rhythms has been associated with aging and disease. Therefore, we hypothesized that individuals with a familial predisposition for longevity have a higher amplitude circadian serum cholesterol concentration rhythm. The aim of this study was to investigate circadian rhythmicity of serum cholesterol concentrations in offspring of nonagenarian siblings and their partners. Offspring from nonagenarian siblings (n = 19), and their partners as controls (n = 18), were recruited from the Leiden Longevity Study. Participants (mean age 65 years) were studied in a controlled in‐hospital setting over a 24‐h period, receiving three isocaloric meals at 9:00 h, 12:00 h and 18:00 h. Lights were off between 23:00 h and 8:00 h. Serum total cholesterol (TC), HDL cholesterol (HDL‐C), non‐HDL‐C and triglycerides (TG) were determined every 30 min over a 24‐h period. Serum TC concentrations were higher during day than during night in offspring (5.2 vs. 4.7 mm , P < 0.001) and in controls (5.3 vs. 5.0 mm , P < 0.001). The difference in TC concentrations between day and night tended to be greater in offspring than in controls (0.5 vs. 0.3 mm , P = 0.109), reaching statistical significance in females (P = 0.045). Notably, the day–night serum differences in non‐HDL‐C were twofold greater in offspring than in controls (0.43 vs. 0.21 mm , P = 0.044) and most explicit in females (0.53 vs. 0.22, P = 0.078). We conclude that familial longevity is characterized by a high circadian rhythmicity of non‐HDL‐C in healthy elderly offspring from nonagenarian siblings.     

Are reproductive and somatic senescence coupled in humans? Late, but not early, reproduction correlated with longevity in historical Sami women

Evolutionary theory of senescence emphasizes the importance of intense selection on early reproduction owing to the declining force of natural selection with age that constrains lifespan. In humans, recent studies have, however, suggested that late-life mortality might be more closely related to late rather than early reproduction, although the role of late reproduction on fitness remains unclear. We examined the association between early and late reproduction with longevity in historical post-reproductive Sami women. We also estimated the strength of natural selection on early and late reproduction using path analysis, and the effect of reproductive timing on offspring survival to adulthood and maternal risk of dying at childbirth. We found that natural selection favoured both earlier start and later cessation of reproduction, and higher total fecundity. Maternal age at childbirth was not related to offspring or maternal survival. Interestingly, females who produced their last offspring at advanced age also lived longest, while age at first reproduction and total fecundity were unrelated to female longevity. Our results thus suggest that reproductive and somatic senescence may have been coupled in these human populations, and that selection could have favoured late reproduction. We discuss alternative hypotheses for the mechanisms which might have promoted the association between late reproduction and longevity.     

Genome-wide association study identifies a single major locus contributing to survival into old age; the APOE locus revisited

By studying the loci that contribute to human longevity, we aim to identify mechanisms that contribute to healthy aging. To identify such loci, we performed a genome-wide association study (GWAS) comparing 403 unrelated nonagenarians from long-living families included in the Leiden Longevity Study (LLS) and 1670 younger population controls. The strongest candidate SNPs from this GWAS have been analyzed in a meta-analysis of nonagenarian cases from the Rotterdam Study, Leiden 85-plus study, and Danish 1905 cohort. Only one of the 62 prioritized SNPs from the GWAS analysis (P<1×10(-4) ) showed genome-wide significance with survival into old age in the meta-analysis of 4149 nonagenarian cases and 7582 younger controls [OR=0.71 (95% CI 0.65-0.77), P=3.39 × 10(-17) ]. This SNP, rs2075650, is located in TOMM40 at chromosome 19q13.32 close to the apolipoprotein E (APOE) gene. Although there was only moderate linkage disequilibrium between rs2075650 and the ApoE ε4 defining SNP rs429358, we could not find an APOE-independent effect of rs2075650 on longevity, either in cross-sectional or in longitudinal analyses. As expected, rs429358 associated with metabolic phenotypes in the offspring of the nonagenarian cases from the LLS and their partners. In addition, we observed a novel association between this locus and serum levels of IGF-1 in women (P=0.005). In conclusion, the major locus determining familial longevity up to high age as detected by GWAS was marked by rs2075650, which tags the deleterious effects of the ApoE ε4 allele. No other major longevity locus was found.     

Differences in the metabolic response to exogenous homocysteine in juvenile and adult rabbits

Homocysteine has recently received a lot of attention as an independent risk factor for atherosclerotic and thrombotic cardiovascular disease. Plasma homocysteine levels tend to rise with age, but are also greatly influenced by nutritional factors. Early reports suggested that there were differences in the metabolism of homocysteine in adult and immature animals. The current work tests the hypothesis that adult and juvenile animals respond differently to chronic administration of homocysteine. We have previously found that adult rabbits given homocysteine parenterally twice daily for seven weeks developed progressive folate deficiency and concurrently developed an impairment of homocysteine metabolism. We now report that juvenile rabbits do not develop folate deficiency with chronic homocysteine loading and do not have progressively higher trough levels of homocysteine, as do the adults. In addition, juvenile rabbits that have been chronically pre-treated with homocysteine exhibit a lower peak homocysteine level after a single dose than do juvenile rabbits that have never received homocysteine. This adaptation did not occur in the adult rabbits. In addition, adult homocysteine-treated rabbits had evidence of oxidative stress as evidenced by higher levels of malondialdehyde in liver tissue than adult controls. The homocysteine-treated juvenile rabbits had the same levels of malondialdehyde as the juvenile control rabbits. We conclude that the plasma elimination kinetics are altered in juvenile rabbits in response to homocysteine pre-treatment. The difference in metabolism of homocysteine may protect the juvenile rabbits from the damaging effects of homocysteine. Future studies are planned to elucidate the mechanism of this adaptive response.     

Decreased Levels of Bisecting GlcNAc Glycoforms of IgG Are Associated with Human Longevity

Background

Markers for longevity that reflect the health condition and predict healthy aging are extremely scarce. Such markers are, however, valuable in aging research. It has been shown previously that the N-glycosylation pattern of human immunoglobulin G (IgG) is age-dependent. Here we investigate whether N-linked glycans reflect early features of human longevity.

Methodology/Principal Findings

The Leiden Longevity Study (LLS) consists of nonagenarian sibling pairs, their offspring, and partners of the offspring serving as control. IgG subclass specific glycosylation patterns were obtained from 1967 participants in the LLS by MALDI-TOF-MS analysis of tryptic IgG Fc glycopeptides. Several regression strategies were applied to evaluate the association of IgG glycosylation with age, sex, and longevity. The degree of galactosylation of IgG decreased with increasing age. For the galactosylated glycoforms the incidence of bisecting GlcNAc increased as a function of age. Sex-related differences were observed at ages below 60 years. Compared to males, younger females had higher galactosylation, which decreased stronger with increasing age, resulting in similar galactosylation for both sexes from 60 onwards. In younger participants (<60 years of age), but not in the older age group (>60 years), decreased levels of non-galactosylated glycoforms containing a bisecting GlcNAc reflected early features of longevity.

Conclusions/Significance

We here describe IgG glycoforms associated with calendar age at all ages and the propensity for longevity before middle age. As modulation of IgG effector functions has been described for various IgG glycosylation features, a modulatory effect may be expected for the longevity marker described in this study.     

Hormonal correlates of dominance in meerkats (Suricata suricatta)

In cooperatively breeding meerkats (Suricata suricatta), individuals typically live in extended family groups in which the dominant male and female are the primary reproductives, while their offspring delay dispersal, seldom breed, and contribute to the care of subsequent litters. Here we investigate hormonal differences between dominants and subordinates by comparing plasma levels of luteinizing hormone (LH), estradiol and cortisol in females, and testosterone and cortisol in males, while controlling for potential confounding factors. In both sexes, hormone levels are correlated with age. In females, levels of sex hormone also vary with body weight and access to unrelated breeding partners in the same group: subordinates in groups containing unrelated males have higher levels of LH and estradiol than those in groups containing related males only. When these effects are controlled, there are no rank-related differences in circulating levels of LH among females or testosterone among males. However, dominant females show higher levels of circulating estradiol than subordinates. Dominant males and females also have significantly higher cortisol levels than subordinates. Hence, we found no evidence that the lower levels of plasma estradiol in subordinate females were associated with high levels of glucocorticoids. These results indicate that future studies need to control for the potentially confounding effects of age, body weight, and access to unrelated breeding partners before concluding that there are fundamental physiological differences between dominant and subordinate group members.     

Hypertension in spontaneously hypertensive rats occurs despite low plasma levels of homocysteine

Hyperhomocysteinemia has been suggested to induce hypertension due to its role in endothelial dysfunction. However, it remains controversial whether homocysteine and hypertension are truly causally related or merely loosely associated. To test the hypothesis that hyperhomocysteinemia occurs in spontaneously hypertensive rats (SHR) we measured plasma levels of homocysteine in 10 male adult SHR and in 10 normotensive controls using ion exchange chromatography. In addition, plasma concentrations of the 22 most common amino acids were measured to explore the relation of homocysteine with amino acid metabolism. Plasma levels of homocysteine were significantly lower in SHR (4.1+/-0.1 micromol/l) than in controls (7.2+/-0.3 micromol/l) (p<0.00001). The amounts of aminobutyric acid, alanine, citrulline and valine were also decreased, whereas we found increased levels of aspartate, glutamate, glutamine, histidine and ornithine. Thus, contrary to our hypothesis, hypertension in SHR occurs despite low plasma levels of homocysteine. We provide a new hypothesis whereby reduced conversion of arginine to citrulline is related to increased ornithine levels, but decreased bioavailability of nitric oxide, resulting in impaired blood vessel relaxation and hypertension. In conclusion, our findings do not necessarily exclude that homocysteine and hypertension might be pathophysiologically connected, but corroborate the notion that hypertension can arise due to mechanisms independent of high homocysteine levels.     

Lipoprotein genotype and conserved pathway for exceptional longevity in humans

Alteration of single genes involved in nutrient and lipoprotein metabolism increases longevity in several animal models. Because exceptional longevity in humans is familial, it is likely that polymorphisms in genes favorably influence certain phenotypes and increase the likelihood of exceptional longevity. A group of Ashkenazi Jewish centenarians ( n = 213), their offspring ( n = 216), and an age-matched Ashkenazi control group ( n = 258) were genotyped for 66 polymorphisms in 36 candidate genes related to cardiovascular disease (CVD). These genes were tested for association with serum lipoprotein levels and particle sizes, apolipoprotein A1, B, and C-3 levels and with outcomes of hypertension, insulin resistance, and mortality. The prevalence of homozygosity for the −641C allele in the APOC3 promoter (rs2542052) was higher in centenarians (25%) and their offspring (20%) than in controls (10%) ( p = 0.0001 and p = 0.001, respectively). This genotype was associated with significantly lower serum levels of APOC3 and a favorable pattern of lipoprotein levels and sizes. We found a lower prevalence of hypertension and greater insulin sensitivity in the −641C homozygotes, suggesting a protective effect against CVD and the metabolic syndrome. Finally, in a prospectively studied cohort, a significant survival advantage was demonstrated in those with the favorable −641C homozygote ( p < 0.0001). Homozygosity for the APOC3 −641C allele is associated with a favorable lipoprotein profile, cardiovascular health, insulin sensitivity, and longevity. Because modulation of lipoproteins is also seen in genetically altered longevity models, it may be a common pathway influencing lifespan from nematodes to humans.     

The role of parental age effects on the evolution of aging

Many studies have found that older parents have shorter-lived offspring. However, the evolutionary significance of these findings is poorly understood. We carried out large-scale demographic experiments to examine the direct effect of maternal age and paternal age on offspring aging in inbred and outbred strains of the fruit fly Drosophila melanogaster. We found that the age of mothers and, to a lesser extent, the age of fathers can have a large influence on both offspring longevity and the shape of the age-specific mortality trajectory. In two independent experiments we found that older mothers generally produced shorter-lived offspring, although the exact effect of maternal age on offspring longevity differed among strains. These results suggest that maternal age effects on progeny aging may influence the evolution of aging.