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Thyroid hormones have vital roles in development, growth and energy metabolism. Within the past two decades, disturbances in thyroid hormone action have been implicated in ageing and the development of age-related diseases. This Review will consider results from biomedical studies that have identified the importance of precise temporospatial regulation of thyroid hormone action for local tissue maintenance and repair. Age-related disturbances in the maintenance of tissue homeostasis are thought to be important drivers of age-related disease. In most iodine-proficient human populations without thyroid disease, the mean, median and 97.5 centile for circulating concentrations of thyroid-stimulating hormone are progressively higher in adults over 80 years of age compared with middle-aged (50-59 years) and younger (20-29 years) adults. This trend has been shown to extend into advanced ages (over 100 years). Here, potential causes and consequences of the altered thyroid status observed in old age and its association with longevity will be discussed. In about 5-20% of adults at least 65 years of age, thyroid-stimulating hormone concentrations are elevated but circulating concentrations of thyroid hormone are within the population reference range, a condition referred to as subclinical hypothyroidism. Results from randomized clinical trials that have tested the clinical benefit of thyroid hormone replacement therapy in older adults with mild subclinical hypothyroidism will be discussed, as well as the implications of these findings for screening and treatment of subclinical hypothyroidism in older adults., (© 2023. Springer Nature Limited.)

Aging is associated with changes in heart rate (HR), heart rate variability (HRV), and 24-h rhythms in HR. Longevity has been linked to lower resting HR, while a higher resting HR and a decreased HRV were linked to cardiovascular events and increased mortality risk. HR and HRV are often investigated during a short electrocardiogram (ECG) measurement at a hospital. In this study, we aim to investigate the relationship between HR parameters with familial longevity and chronological age derived from continuous ambulatory ECG measurements collected over a period of 24 to 90 hours. We included 73 middle-aged participants (mean (SD) age: 67.0 (6.16) years), comprising 37 offspring of long-lived families, 36 of their partners, and 35 young participants (22.8 (3.96) years). We found no association with familial longevity, but middle-aged participants had lower 24-h HR (average and maximum HR, not minimum HR), lower amplitudes, and earlier trough and peak times than young participants. Associations in HR with chronological age could be caused by the aging process or by differences in environmental factors. Interestingly, middle-aged participants had a less optimal HRV during long-term recordings in both the sleep and awake periods, which might indicate that their heart is less adaptable than that of young participants. This could be a first indication of deteriorated cardiovascular health in middle-aged individuals.

Context: Offspring from long-lived families have a different thyroid status than controls, characterised by higher circulating levels of thyroid stimulating hormone (TSH) and similar levels of thyroid hormone. Expression of the TSH receptor has previously been observed on various extrathyroidal tissues, including bone. However, potential physiological consequences of differences in circulating TSH as observed in familial longevity on bone tissue remain unclear., Objective: Based on the hypothesis that TSH may inhibit bone resorption, we explored whether offspring of long-lived families have lower bone turnover than controls at baseline as well as following a challenge with recombinant human TSH (rhTSH)., Methods: Bone turnover markers CTX and P1NP were measured in fasted morning samples from 14 offspring and 12 controls at baseline and at 24 hour intervals following 0.1 mg rhTSH i.m. administration for four consecutive days., Results: At baseline, mean (SEM) CTX was 0.32 (0.03) ng/ml in offspring and 0.50 (0.04) ng/ml in controls, p < 0.01, whereas mean (SEM) P1NP was 39.6 (3.2) ng/ml in offspring and 61.8 (6.6) ng/ml in controls, p < 0.01. Following rhTSH administration, both CTX and P1NP levels transiently increased over time and normalized towards baseline after 72 h (general linear modelling: CTX time p = 0.01, P1NP time p < 0.01); the response was similar between offspring and controls., Conclusions: Bone turnover markers were lower at baseline in offspring from long-lived families than in controls but increased similarly following an rhTSH challenge.

Context: Longevity is associated with higher circulating levels of TSH in the absence of differences in circulating thyroid hormones (TH), as previously observed in F2 members of long-lived families (F2-LLS) and their partners (F2-Con). The mechanism underlying this observed difference remains unknown., Objective: We hypothesized that the thyroid gland of members from long-lived families are less responsive to TSH stimulation, thereby requiring higher circulating TSH levels to maintain adequate TH levels., Methods: We performed a case-control intervention study with a single intramuscular (gluteal) injection with 0.1 mg recombinant human TSH in a subgroup of 14 F2-LLS and 15 similarly aged F2-Con. They were followed for 4 days. No serious adverse events were reported. For analyses, we compared time trajectories of TSH and TH, and the ratio of TH to TSH using area under the curve (AUC) calculations., Results: The AUC free T4/AUC TSH ratio was significantly lower in F2-LLS than in F2-Con (estimated mean [95% confidence interval] 1.6 [1.2-1.9] and 2.2 [1.9-2.6], respectively, P = 0.01). The AUC thyroglobulin/AUC TSH ratio was also lower in F2-LLS than in F2-Con (median [interquartile range] 2.1 [1.4-3.6] and 3.2 [2.7-7.4], respectively, P = 0.04). We observed the same trend with the AUC free T3/AUC TSH ratio, although the difference was not statistically significant (estimated mean [95% confidence interval] 0.6 [0.4-0.7] and 0.7 [0.6-0.8], respectively, P = 0.07)., Conclusions: The present findings show that members of long-living families have a lower thyroid responsivity to TSH compared with their partners., (© Endocrine Society 2020.)

Context: Hormones of the hypothalamic-pituitary-target gland axes are mostly investigated separately, whereas the interplay between hormones might be as important as each separate hormonal axis., Objective: Our aim is to determine the interrelationships between GH, TSH, ACTH, and cortisol in healthy older individuals., Design: We made use of 24-hour hormone serum concentrations assessed with intervals of 10 minutes from 38 healthy older individuals with a mean age (SD) of 65.1 (5.1) years from the Leiden Longevity Study. Cross-correlation analyses were performed to assess the relative strength between 2 24-hour hormone serum concentration series for all possible time shifts. Cross-approximate entropy was used to assess pattern synchronicity between 2 24-hour hormone serum concentration series., Results: Within an interlinked hormonal axis, ACTH and cortisol were positively correlated with a mean (95% confidence interval) correlation coefficient of 0.78 (0.74-0.81) with cortisol following ACTH concentrations with a delay of 10 minutes. Between different hormonal axes, we observed a negative correlation coefficient between cortisol and TSH of -0.30 (-0.36 to -0.25) with TSH following cortisol concentrations with a delay of 170 minutes. Furthermore, a positive mean (95% confidence interval) correlation coefficient of 0.29 (0.22-0.37) was found between TSH and GH concentrations without any delay. Moreover, cross-approximate entropy analyses showed that GH and cortisol exhibit synchronous serum concentration patterns., Conclusions: This study demonstrates that interrelations between hormones from interlinked as well as different hypothalamic-pituitary-target gland axes are observed in healthy older individuals. More research is needed to determine the biological meaning and clinical consequences of these observations., (© Endocrine Society 2019.)

Human longevity is heritable, but genome-wide association (GWA) studies have had limited success. Here, we perform two meta-analyses of GWA studies of a rigorous longevity phenotype definition including 11,262/3484 cases surviving at or beyond the age corresponding to the 90th/99th survival percentile, respectively, and 25,483 controls whose age at death or at last contact was at or below the age corresponding to the 60th survival percentile. Consistent with previous reports, rs429358 (apolipoprotein E (ApoE) ε4) is associated with lower odds of surviving to the 90th and 99th percentile age, while rs7412 (ApoE ε2) shows the opposite. Moreover, rs7676745, located near GPR78, associates with lower odds of surviving to the 90th percentile age. Gene-level association analysis reveals a role for tissue-specific expression of multiple genes in longevity. Finally, genetic correlation of the longevity GWA results with that of several disease-related phenotypes points to a shared genetic architecture between health and longevity.

The relationship between thyroid status and longevity has been investigated extensively. However, data on thyroid status and survival in old age is scarce. In this study we investigated associations of different parameters of thyroid status with mortality in nonagenarians, and whether these associations were different in nonagenarians from long-lived families than in nonagenarians from the general population. In total, 805 nonagenarians from the Leiden Longevity Study and 259 nonagenarians from the Leiden 85-plus Study were followed up to collect mortality data. At baseline, levels of thyrotropin (TSH), free thyroxine (fT4) and free triiodothyronine (fT3) were measured. In nonagenarians from long-lived families and from the general population, associations between thyroid parameters and mortality were similar. We found no interaction between study population and parameters of thyroid status on mortality (P-values>0.70). The results from both studies were combined to derive generalizable associations. Hazard ratios (HRs) for the highest compared to lowest tertiles were determined, resulting in TSH HR 0.91 (P=0.25), fT4 HR 1.22 (P=0.02), fT3 HR 0.74 (P=1.31e-4), and fT3/fT4 HR 0.66 (P=5.64e-7). In conclusion, higher fT3/fT4 ratios, higher levels of fT3, and lower levels of fT4 were associated with lower mortality rate in nonagenarians and independent of familial longevity status.

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., (© 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Genetic studies have thus far identified a limited number of loci associated with human longevity by applying age at death or survival up to advanced ages as phenotype. As an alternative approach, one could first try to identify biomarkers of healthy ageing and the genetic variants associated with these traits and subsequently determine the association of these variants with human longevity. In the present study, we used this approach by testing whether the 35 baseline serum parameters measured in the Leiden Longevity Study (LLS) meet the proposed criteria for a biomarker of healthy ageing. The LLS consists of 421 families with long-lived siblings of European descent, who were recruited together with their offspring and the spouses of the offspring (controls). To test the four criteria for a biomarker of healthy ageing in the LLS, we determined the association of the serum parameters with chronological age, familial longevity, general practitioner-reported general health, and mortality. Out of the 35 serum parameters, we identified glucose, insulin, and triglycerides as biomarkers of healthy ageing, meeting all four criteria in the LLS. We subsequently showed that the genetic variants previously associated with these parameters are significantly enriched in the largest genome-wide association study for human longevity. In conclusion, we showed that biomarkers of healthy ageing can be used to leverage genetic studies into human longevity. We identified several genetic variants influencing the variation in glucose, insulin and triglycerides that contribute to human longevity., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Offspring of long-lived families are characterized by beneficial metabolic phenotypes in glucose and lipid metabolism and low 25-hydroxyvitamin D. Although the genetic basis for human longevity remains largely unclear, the contribution of variation at the APOE locus has been repeatedly demonstrated. We aimed to assess whether ApoE isoforms mark the familial longevity status in middle age and subsequently to test to what extend this association is mediated by the metabolic characteristics marking this status. From the Leiden Longevity Study (LLS), we included offspring from nonagenarian siblings and partners as controls. Using the metabolic phenotypes of familial longevity as mediators, we investigated how APOE gene variants associated with LLS offspring/control status (in 1,515 LLS offspring and 715 controls). Within the LLS (mean age = 59.2 years), ApoE ε4 was not associated with a lower likelihood of being an LLS offspring, whereas ApoE ɛ2 was significantly associated with a higher likelihood of being an LLS offspring (odds ratio = 1.43), but this difference was not mediated (p-values>0.05) by any of the investigated metabolic phenotypes (e.g., diabetes and glucose). Therefore, variation at the APOE locus may not influence familial longevity status in middle age significantly through any of the metabolic mechanisms investigated., Competing Interests: All authors declare to have no conflict of interest.

Individuals enriched for familial longevity display a lower prevalence of age-related diseases, such as cardiovascular- and metabolic diseases. Since these diseases are associated with stress and increased cortisol levels, one of the underlying mechanisms that may contribute to healthy longevity might be a more adaptive response to stress. To investigate this, male middle-aged offspring from long-lived families (n = 31) and male non-offspring (with no familial history of longevity) (n = 26) were randomly allocated to the Trier Social Stress Test or a control condition in an experimental design. Physiological (cortisol, blood pressure, heart rate) and subjective responses were measured during the entire procedure. The results showed that Offspring had lower overall cortisol levels compared to Non-offspring regardless of condition, and lower absolute cortisol output (AUCg) during stress compared to Non-Offspring, while the increase (AUCi) did not differ between groups. In addition, systolic blood pressure in Offspring was lower compared to Non-offspring during the entire procedure. At baseline, Offspring had significantly lower systolic blood pressure and reported less subjective stress than Non-offspring and showed a trend towards lower heart rate. Offspring from long-lived families might thus be less stressed prior to potentially stressful events and consequently show overall lower levels in physiological responses. Although attenuated physiological responding cannot be ruled out, lower starting points and a lower peak level in physiological responding when confronted with an actual stressor, might already limit damage due to stress over a lifetime. Lower physiological responding may also contribute to the lower prevalence of cardiovascular diseases and other stress-related diseases in healthy longevity.

Reduced insulin/insulin-like growth factor 1 (IGF-1) signaling has been associated with longevity in various model organisms. However, the role of insulin/IGF-1 signaling in human survival remains controversial. The aim of this study was to test whether circulating IGF-1 axis parameters associate with old age survival and functional status in nonagenarians from the Leiden Longevity Study. This study examined 858 Dutch nonagenarian (males≥89 years; females≥91 years) siblings from 409 families, without selection on health or demographic characteristics. Nonagenarians were divided over sex-specific strata according to their levels of IGF-1, IGF binding protein 3 and IGF-1/IGFBP3 molar ratio. We found that lower IGF-1/IGFBP3 ratios were associated with improved survival: nonagenarians in the quartile of the lowest ratio had a lower estimated hazard ratio (95% confidence interval) of 0.73 (0.59 - 0.91) compared to the quartile with the highest ratio (ptrend=0.002). Functional status was assessed by (Instrumental) Activities of Daily Living ((I)ADL) scales. Compared to those in the quartile with the highest IGF-1/IGFBP3 ratio, nonagenarians in the lowest quartile had higher scores for ADL (ptrend=0.001) and IADL (ptrend=0.003). These findings suggest that IGF-1 axis parameters are associated with increased old age survival and better functional status in nonagenarians from the Leiden Longevity Study.

Context: Longevity is associated with changes in circulating levels of thyroid hormone (TH) and/or TSH in animals and humans, but underlying mechanisms remain elusive., Objective: We explored in 38 offspring of nonagenarian participants from the Leiden Longevity Study, who are enriched for longevity and in their partners, ultradian and circadian rhythmicity of TSH, temporal relationship, and feedback and forward interplay between TSH and TH., Methods: We collected blood samples every 10 minutes for 24 hours for TSH and TH profiles. We used a deconvolution analysis to estimate basal (nonpulsatile), pulsatile, and other secretion parameters to characterize ultradian rhythmicity and locally weighted polynomial regression of TSH to assess circadian rhythmicity. A cross-correlation analysis was used to investigate the temporal relationship between TSH and TH and cross-approximate entropy to assess feedback and forward interplay between TSH and TH., Results: Compared with partners, offspring displayed higher mean (95% confidence interval [CI]) basal TSH secretion (34.3 [95% CI 27.2-43.1] mU/L per 24 hours vs 18.5 [95% CI 14.4-23.7] mU/L per 24 hours, P = .001) but no differences in ultradian or circadian properties of TSH. The temporal relationship between TSH and free T3 at zero delay was higher in offspring (0.48 ± 0.2) compared with partners (0.26 ± 0.4) (P = .05), but the feedback and forward interplay between TSH and TH did not differ., Conclusions: Familial longevity is associated with increased basal TSH secretion and a strong temporal relationship between TSH and free T3 but not with differences in ultradian or circadian TSH rhythmicity or feedback and forward interplay between TSH and TH.

Objective: The hypothalamic-pituitary-adrenal (HPA)-axis is the most important neuro-endocrine stress response system of our body which is of critical importance for survival. Disturbances in HPA-axis activity have been associated with adverse metabolic and cognitive changes. Humans enriched for longevity have less metabolic and cognitive disturbances and therefore diminished activity of the HPA axis may be a potential candidate mechanism underlying healthy familial longevity. Here, we compared 24-h plasma ACTH and serum cortisol concentration profiles and different aspects of the regulation of the HPA-axis in offspring from long-lived siblings, who are enriched for familial longevity and age-matched controls., Design: Case-control study within the Leiden Longevity study cohort consisting of 20 middle-aged offspring of nonagenarian siblings (offspring) together with 18 partners (controls)., Methods: During 24 h, venous blood was sampled every 10 minutes for determination of circulatory ACTH and cortisol concentrations. Deconvolution analysis, cross approximate entropy analysis and ACTH-cortisol-dose response modeling were used to assess, respectively, ACTH and cortisol secretion parameters, feedforward and feedback synchrony and adrenal gland ACTH responsivity., Results: Mean (95% Confidence Interval) basal ACTH secretion was higher in male offspring compared to male controls (645 (324-1286) ngl/L/24 h versus 240 (120-477) ng/L/24 h, P = 0.05). Other ACTH and cortisol secretion parameters did not differ between offspring and controls. In addition, no significant differences in feedforward and feedback synchrony and adrenal gland ACTH responsivity were observed between groups., Conclusions: These results suggest that familial longevity is not associated with major differences in HPA-axis activity under resting conditions, although modest, sex-specific differences may exist between groups that might be clinically relevant.

Few studies have included subjects with the propensity to reach old age in good health, with the aim to disentangle mechanisms contributing to staying healthier for longer. The hypothalamic-pituitary-thyroid (HPT) axis maintains circulating levels of thyroid stimulating hormone (TSH) and thyroid hormone (TH) in an inverse relationship. Greater longevity has been associated with higher TSH and lower TH levels, but mechanisms underlying TSH/TH differences and longevity remain unknown. The HPT axis plays a pivotal role in growth, development and energy metabolism. We report that offspring of nonagenarians with at least one nonagenarian sibling have increased TSH secretion but similar bioactivity of TSH and similar TH levels compared to controls. Healthy offspring and spousal controls had similar resting metabolic rate and core body temperature. We propose that pleiotropic effects of the HPT axis may favour longevity without altering energy metabolism.

The genetic contribution to the variation in human lifespan is ∼ 25%. Despite the large number of identified disease-susceptibility loci, it is not known which loci influence population mortality. We performed a genome-wide association meta-analysis of 7729 long-lived individuals of European descent (≥ 85 years) and 16 121 younger controls (<65 years) followed by replication in an additional set of 13 060 long-lived individuals and 61 156 controls. In addition, we performed a subset analysis in cases aged ≥ 90 years. We observed genome-wide significant association with longevity, as reflected by survival to ages beyond 90 years, at a novel locus, rs2149954, on chromosome 5q33.3 (OR = 1.10, P = 1.74 × 10(-8)). We also confirmed association of rs4420638 on chromosome 19q13.32 (OR = 0.72, P = 3.40 × 10(-36)), representing the TOMM40/APOE/APOC1 locus. In a prospective meta-analysis (n = 34 103), the minor allele of rs2149954 (T) on chromosome 5q33.3 associates with increased survival (HR = 0.95, P = 0.003). This allele has previously been reported to associate with low blood pressure in middle age. Interestingly, the minor allele (T) associates with decreased cardiovascular mortality risk, independent of blood pressure. We report on the first GWAS-identified longevity locus on chromosome 5q33.3 influencing survival in the general European population. The minor allele of this locus associates with low blood pressure in middle age, although the contribution of this allele to survival may be less dependent on blood pressure. Hence, the pleiotropic mechanisms by which this intragenic variation contributes to lifespan regulation have to be elucidated., (© The Author 2014. Published by Oxford University Press.)

Objective: Familial longevity is marked by enhanced peripheral but not hepatic insulin sensitivity. The liver has a critical role in the pathogenesis of hepatic insulin resistance. Therefore we hypothesized that the extent of liver steatosis would be similar between offspring of long-lived siblings and control subjects. To test our hypothesis, we investigated the extent of liver steatosis in non-diabetic offspring of long-lived siblings and age-matched controls by measuring liver enzymes in plasma and liver fat by computed tomography (CT)., Research Design and Methods: We measured nonfasting alanine transaminase (ALT), aspartate aminotransferase (AST), and Υ-glutamyl transferase (GGT) in 1625 subjects (736 men, mean age 59.1 years) from the Leiden Longevity Study, comprising offspring of long-lived siblings and partners thereof. In a random subgroup, fasting serum samples (n = 230) were evaluated and CT was performed (n = 268) for assessment of liver-spleen (L/S) ratio and the prevalence of moderate-to-severe non-alcoholic fatty liver disease (NAFLD). Linear mixed model analysis was performed adjusting for age, gender, body mass index, smoking, use of alcohol and hepatotoxic medication, and correlation of sibling relationship., Results: Offspring of long-lived siblings had higher nonfasting ALT levels as compared to control subjects (24.3 mmol/L versus 23.2 mmol/L, p = 0.03), while AST and GGT levels were similar between the two groups. All fasting liver enzyme levels were similar between the two groups. CT L/S ratio and prevalence of moderate-to-severe NAFLD was similar between groups (1.12 vs 1.14, p = 0.25 and 8% versus 8%, p = 0.91, respectively)., Conclusions: Except for nonfasting levels of ALT, which were slightly higher in the offspring of long-lived siblings compared to controls, no differences were found between groups in the extent of liver steatosis, as assessed with liver biochemical tests and CT. Thus, our data indicate that the extent of liver steatosis is similar between offspring of long-lived siblings and control subjects.

Studying renal function in subjects with a familial propensity for longevity may provide insight in (un)known mechanisms that determine the age-related decline in renal function of normal subjects. In the Leiden Longevity Study, middle-aged offspring of non-agenarian siblings and their partners as environmentally matched controls were included. Information was collected on lifestyle, medical history, medication use, and a non-fasting blood sample was drawn. Renal function (estimated glomerular filtration rate, eGFR) was assessed with the Chronic Kidney Disease epidemiology collaboration (CKD-EPI) formula. Linear mixed models were used to account for familial dependencies within the offspring and all analyses were stratified by sex. eGFR was similar between female offspring and female controls (0.44ml/min/1.73m(2) (SE 0.72) difference, p=0.54, age-adjusted). Male offspring had a higher eGFR compared to male controls (1.78ml/min/1.73m(2) (SE 0.78) difference, p=0.022, age-adjusted), and further adjustments for various characteristics did not materially change this difference. Among men with a history of hypertension, or myocardial infarction and/or stroke, offspring had a higher eGFR compared to controls (4.74ml/min/1.73m(2) (SE 1.53) difference, p=0.002, age-adjusted, and 6.21ml/min/1.73m(2) (SE 2.85) difference, p=0.033, age-adjusted, respectively). Middle-aged men, but not women, with a propensity for longevity have better renal function compared to environmentally matched controls, especially among those with a history of cardiovascular disease., (Copyright © 2013 Elsevier Inc. All rights reserved.)

mTOR signalling is implicated in the development of disease and in lifespan extension in model organisms. This pathway has been associated with human diseases such as diabetes and cancer, but has not been investigated for its impact on longevity per se. Here, we investigated whether transcriptional variation within the mTOR pathway is associated with human longevity using whole-blood samples from the Leiden Longevity Study. This is a unique cohort of Dutch families with extended survival across generations, decreased morbidity and beneficial metabolic profiles in middle-age. By comparing mRNA levels of nonagenarians and middle-aged controls, the mTOR signalling gene set was found to associate with old age (P = 4.6 × 10(-7)). Single gene analysis showed that seven of 40 mTOR pathway genes had a significant differential expression of at least 5%. Of these, the RPTOR (Raptor) gene was found to be differentially expressed also when the offspring of nonagenarians was compared with their spouses, indicating association with familial longevity in middle-age. This association was not explained by variation between the groups in the prevalence of type 2 diabetes and cancer or glucose levels. Thus, the mTOR pathway not only plays a role in the regulation of disease and aging in animal models, but also in human health and longevity., (© 2012 The Authors Aging Cell © 2012 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.)

In genome-wide association studies (GWAS) of complex traits, single SNP analysis is still the most applied approach. However, the identified SNPs have small effects and provide limited biological insight. A more appropriate approach to interpret GWAS data of complex traits is to analyze the combined effect of a SNP set grouped per pathway or gene region. We used this approach to study the joint effect on human longevity of genetic variation in two candidate pathways, the insulin/insulin-like growth factor (IGF-1) signaling (IIS) pathway and the telomere maintenance (TM) pathway. For the analyses, we used genotyped GWAS data of 403 unrelated nonagenarians from long-lived sibships collected in the Leiden Longevity Study and 1,670 younger population controls. We analyzed 1,021 SNPs in 68 IIS pathway genes and 88 SNPs in 13 TM pathway genes using four self-contained pathway tests (PLINK set-based test, Global test, GRASS and SNP ratio test). Although we observed small differences between the results of the different pathway tests, they showed consistent significant association of the IIS and TM pathway SNP sets with longevity. Analysis of gene SNP sets from these pathways indicates that the association of the IIS pathway is scattered over several genes (AKT1, AKT3, FOXO4, IGF2, INS, PIK3CA, SGK, SGK2, and YWHAG), while the association of the TM pathway seems to be mainly determined by one gene (POT1). In conclusion, this study shows that genetic variation in genes involved in the IIS and TM pathways is associated with human longevity.

Estimating perceived age by facial photographs is a good estimate of health in elderly populations. Previously, we showed that familial longevity is marked by a more beneficial glucose metabolism already at middle age. As glucose is also related to skin aging, this study aimed to investigate the association between glucose metabolism and perceived age. Perceived age was assessed using facial photographs and non-fasted glucose and insulin were measured in 602 subjects from the Leiden Longevity Study. Non-diabetic subjects (n = 569) were divided in three strata according to their glucose levels, and diabetic subjects (n = 33; as a proxy of long-term hyperglycemic exposure) were included as a fourth stratum. Considered confounding factors were gender, chronological age, current smoking, body mass index, photo-damage score, and insulin levels. Perceived age was increased from 59.6 years (SE = 0.3) in the first stratum to 61.2 years (SE = 0.6) in diabetic subjects (p for trend = 0.002). In non-diabetic subjects only, perceived age was increased from 59.6 years (SE = 0.3) in the first stratum to 60.6 years (SE = 0.3) in the third stratum (p for trend = 0.009). Continuously, perceived age increased 0.40 years (SE = 0.14, p = 0.006) per 1 mmol/L increase in glucose level in non-diabetic subjects. The present study demonstrates that, also among non-diabetic subjects, higher glucose levels are associated with a higher perceived age. Future research should be focused on elucidating possible mechanisms linking glucose levels to perceived age.

Background: As facial appearance can be readily quantified and skin tissue easily accessed, they could be valuable tools for determining how biological mechanisms influence tissue degeneration with age and, consequently, human health and lifespan. It is unknown, however, whether appearance reflects disease risk or lifespan independently of factors already known to associate with both health and appearance., Methods: In a cross-sectional study, we compared the amount of skin wrinkling on a sun-protected site (upper inner arm) and the facial appearance of 261 offspring (mean age 63.2 y) of nonagenarian siblings with 253 age-matched controls (mean age 62.7 y), all with no reported disease history. We next examined whether any appearance features that significantly associated with familial longevity also associated with the Framingham cardiovascular disease (CVD) risk score. All analyses were adjusted for chronological age, smoking, photodamage, and body mass index., Results: Female and male offspring had reduced upper inner arm skin wrinkling (p = .03 and p < .001, respectively), and the male offspring looked 1.4 y younger than the controls (p = .002). There were no significant associations between CVD risk and upper inner arm skin wrinkling. Women in the lowest quartile of CVD risk looked more than 2 y younger for their age than those in higher risk quartiles (p = .002). Systolic blood pressure was the most significant (p = .004) CVD risk factor that was associated with perceived age in women., Conclusions: Facial appearance and skin wrinkling at a sun-protected site reflect the propensity to reach an extreme old age, and facial appearance reflects the risk of succumbing to CVD independently of chronological age, smoking, photodamage, and BMI.

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.

Background: Low levels of 25(OH) vitamin D are associated with various age-related diseases and mortality, but causality has not been determined. We investigated vitamin D levels in the offspring of nonagenarians who had at least one nonagenarian sibling; these offspring have a lower prevalence of age-related diseases and a higher propensity to reach old age compared with their partners., Methods: We assessed anthropometric characteristics, 25(OH) vitamin D levels, parathyroid hormone levels, dietary vitamin D intake and single nucleotide polymorphisms (SNPs) associated with vitamin D levels. We included offspring (n = 1038) of nonagenarians who had at least one nonagenarian sibling, and the offsprings' partners (n = 461; controls) from the Leiden Longevity Study. We included age, sex, body mass index, month during which blood sampling was performed, dietary and supplemental vitamin D intake, and creatinine levels as possible confounding factors., Results: The offspring had significantly lower levels of vitamin D (64.3 nmol/L) compared with controls (68.4 nmol/L; p = 0.002), independent of possible confounding factors. There was no difference in the levels of parathyroid hormone between groups. Compared with controls, the offspring had a lower frequency of a genetic variant in the CYP2R1 gene (rs2060793) (p = 0.04). The difference in vitamin D levels between offspring and controls persisted over the 2 most prevalent genotypes of this SNP., Interpretation: Compared with controls, the offspring of nonagenarians who had at least one nonagenarian sibling had a reduced frequency of a common variant in the CYP2R1 gene, which predisposes people to high vitamin D levels; they also had lower levels of vitamin D that persisted over the 2 most prevalent genotypes. These results cast doubt on the causal nature of previously reported associations between low levels of vitamin D and age-related diseases and mortality.

The effect of chronological age on skin characteristics is readily visible, and its underlying histological changes have been a field of study for several years. However, the effect of biological age (i.e. a person's rate of ageing compared to their chronological age) on the skin has so far only been studied in facial photographs. Skin biopsies obtained from middle-aged offspring of nonagenarian siblings that are genetically enriched for longevity were compared to their partners who represent the general Dutch population. Though of the same chronological age, the offspring were previously observed to be of a younger biological age than their partners. The biopsies were analysed on several aspects epidermal and elastic fibre morphology. We investigated whether these skin characteristics were dependent on chronological age, familial longevity (the difference between the offspring and partners) and Framingham heart risk scores, adjusted for external stressors. A decreased thickness and flattening of the epidermis as well as an increased amount of elastic fibres in the reticular dermis were observed with chronological age (P < 0.001, P < 0.001 and P = 0.03, respectively), but no effect of familial longevity was found. The Framingham heart risk score was associated with some skin characteristics. A slower rate of skin ageing does not mark offspring from nonagenarian siblings. Epidermal and elastic fibre morphometric characteristics are not a potential marker for familial longevity in middle-aged subjects enriched for familial longevity.

Background: Cortisol levels are strongly associated with a person's health. Familial longevity and age assessment of facial photographs (perceived age) are both associated with morbidity and mortality. The present study aimed to investigate morning cortisol levels in familial longevity and the association of these levels with perceived age., Methods: Perceived age and serum morning cortisol levels were measured for 138 offspring from long-lived families and 138 partners from the Leiden Longevity Study. Considered confounding factors were chronological age, gender, body mass index, current smoking habits, antidepressant drug use, antihypertensive drugs and diabetes medication., Results: In the fully adjusted model, which was restricted to participants who did not use antidepressant drugs, offspring had similar serum cortisol levels compared to their partners (0.54 and 0.55μmol/L, respectively; p=0.54). Using a similar model taking offspring and partners together, an increase of 0.1μmol/L in morning cortisol levels was associated with an 0.42 (95% CI 0.0-0.84, p=0.048) year increase in perceived age. This association was significantly attenuated in the offspring group (0.01, 95% CI -0.58 to 0.59, p=0.98) compared to the partner group (0.81, 95% CI 0.20-1.41, p=0.009 year increase in perceived age per 0.1μmol/L increase in cortisol respectively) (p for interaction=0.042)., Conclusion: This study demonstrates that high levels of cortisol are associated with a higher perceived age. This association was attenuated in offspring from long-lived families compared to their partners, suggesting enhanced stress resistance in these subjects. Future research will be aimed at elucidating potential mechanisms underlying the observations in this study., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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.

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.

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.

Background: Reported findings are inconsistent whether hypothalamic-pituitary-adrenal (HPA) signaling becomes hyperactive with increasing age, resulting in increasing levels of cortisol. Our previous research strongly suggests that offspring from long-lived families are biologically younger. In this study we assessed whether these offspring have a lower HPA axis activity, as measured by lower levels of cortisol and higher cortisol feedback sensitivity., Methods: Salivary cortisol levels were measured at four time points within the first hour upon awakening and at two time points in the evening in a cohort comprising 149 offspring and 154 partners from the Leiden Longevity Study. A dexamethasone suppression test was performed as a measure of cortisol feedback sensitivity. Age, gender and body mass index, smoking and disease history (type 2 diabetes and hypertension) were considered as possible confounding factors., Results: Salivary cortisol secretion was lower in offspring compared to partners in the morning (Area Under the Curve = 15.6 versus 17.1 nmol/L, respectively; p = 0.048) and in the evening (Area Under the Curve = 3.32 versus 3.82 nmol/L, respectively; p = 0.024). Salivary cortisol levels were not different after dexamethasone (0.5 mg) suppression between offspring and partners (4.82 versus 5.26 nmol/L, respectively; p = 0.28)., Conclusion: Offspring of nonagenarian siblings are marked by a lower HPA axis activity (reflected by lower diurnal salivary cortisol levels), but not by a difference in cortisol feedback sensitivity. Further in-depth studies aimed at characterizing the HPA axis in offspring and partners are needed.

Earlier, we showed that the offspring from exceptionally long-lived families have a more favorable glucose metabolism when compared with controls. As chronic low-grade inflammation has been regarded as a strong risk factor for insulin resistance, we evaluated if and to what extent the favorable glucose metabolism in offspring from long-lived families could be explained by differences in subclinical inflammation, as estimated from circulating levels of C-reactive protein. We found no difference between the two groups in C-reactive protein levels or in the distribution of C-reactive protein haplotypes. However, among controls higher levels of C-reactive protein were related to higher glucose levels, whereas among offspring levels of C-reactive protein were unrelated to glucose levels. It is a limitation of the current study that its cross-sectional nature does not allow for assessment of cause-effect relationships. One possible interpretation of these data is that the offspring from long-lived families might be able to regulate glucose levels more tightly under conditions of low-grade inflammation. To test this hypothesis, our future research will be focused on assessing the robustness of insulin sensitivity in response to various challenges in offspring from long-lived families and controls.

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., (© 2011 The Authors. Aging Cell © 2011 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.)

Mechanisms underlying the variation in human life expectancy are largely unknown, but lipid metabolism and especially lipoprotein size was suggested to play an important role in longevity. We have performed comprehensive lipid phenotyping in the Leiden Longevity Study (LLS). By applying multiple logistic regression analysis we tested for the first time the effects of parameters in lipid metabolism (i.e., classical serum lipids, lipoprotein particle sizes, and apolipoprotein E levels) on longevity independent of each other. Parameters in lipid metabolism were measured in offspring of nonagenarian siblings from 421 families of the LLS (n = 1,664; mean age, 59 years) and in the partners of the offspring as population controls (n = 711; mean age, 60 years). In the initial model, where lipoprotein particles sizes, classical serum lipids and apolipoprotein E were included, offspring had larger low-density lipoprotein (LDL) particle sizes (p = 0.017), and lower triglyceride levels (p = 0.026), indicating that they displayed a more beneficial lipid profile. After backwards regression only LDL size (p = 0.014) and triglyceride levels (p = 0.05) were associated with offspring from long-lived families. Sex-specific backwards regression analysis revealed that LDL particle sizes were associated with male longevity (increase in log odds ratio (OR) per unit = 0.21; p = 0.023). Triglyceride levels (decrease OR per unit = 0.22; p = 0.01), but not LDL particle size, were associated with female longevity. Due to the analysis of a comprehensive lipid profile, we confirmed an important role of lipid metabolism in human longevity, with LDL size and triglyceride levels as major predicting factors.

Homocysteine concentrations are a read-out of methionine metabolism and have been related to changes in lifespan in animal models. In humans, high homocysteine concentrations are an important predictor of age related disease. We aimed to explore the association of homocysteine with familial longevity by testing whether homocysteine is lower in individuals that are genetically enriched for longevity. We measured concentrations of total homocysteine in 1907 subjects from the Leiden Longevity Study consisting of 1309 offspring of nonagenarian siblings, who are enriched with familial factors promoting longevity, and 598 partners thereof as population controls. We found that homocysteine was related to age, creatinine, folate, vitamin B levels and medical history of hypertension and stroke in both groups (all p<0.001). However, levels of homocysteine did not differ between offspring enriched for longevity and their partners, and no differences in the age-related rise in homocysteine levels were found between groups (p for interaction 0.63). The results suggest that homocysteine metabolism is not likely to predict familial longevity.

Insulin resistance is a risk factor for various age-related diseases. In the Leiden Longevity study, we recruited long-lived siblings and their offspring. Previously, we showed that, compared to controls, the offspring of long-lived siblings had a better glucose tolerance. Here, we compared groups of offspring from long-lived siblings and controls for the relation between insulin and glucose in nonfasted serum (n = 1848 subjects) and for quantitation of insulin action using a two-step hyperinsulinemic-euglycemic clamp (n = 24 subjects). Groups of offspring and controls were similar with regard to sex distribution, age, and body mass index. We observed a positive bi-phasic linear relationship between ln (insulin) levels and nonfasted glucose with a steeper slope from 10.7mU L(-1) insulin onwards in controls compared to offspring (P = 0.02). During the clamp study, higher glucose infusion rate was required to maintain euglycemia during high-dose insulin infusion (P = 0.036) in offspring, reflecting higher whole-body insulin sensitivity. After adjustment for sex, age, and fat mass, the insulin-mediated glucose disposal rate (GDR) was higher in offspring than controls (42.5 ± 2.7 vs. 33.2 ± 2.7 micromol kg(-1) min(-1) , mean ± SE, P = 0.025). The insulin-mediated suppression of endogenous glucose production and lipolysis did not differ between groups (all P > 0.05). Furthermore, GDR was significantly correlated with the mean age of death of the parents. In conclusion, offspring from long-lived siblings are marked by enhanced peripheral glucose disposal. Future research will focus on identifying the underlying biomolecular mechanisms, with the aim to promote health in old age.

In animal models, single-gene mutations in genes involved in insulin/IGF and target of rapamycin signalling pathways extend lifespan to a considerable extent. The genetic, genomic and epigenetic influences on human longevity are expected to be much more complex. Strikingly however, beneficial metabolic and cellular features of long-lived families resemble those in animals for whom the lifespan is extended by applying genetic manipulation and, especially, dietary restriction. Candidate gene studies in humans support the notion that human orthologues from longevity genes identified in lower species do contribute to longevity but that the influence of the genetic variants involved is small. Here we discuss how an integration of novel study designs, labour-intensive biobanking, deep phenotyping and genomic research may provide insights into the mechanisms that drive human longevity and healthy ageing, beyond the associations usually provided by molecular and genetic epidemiology. Although prospective studies of humans from the cradle to the grave have never been performed, it is feasible to extract life histories from different cohorts jointly covering the molecular changes that occur with age from early development all the way up to the age at death. By the integration of research in different study cohorts, and with research in animal models, biological research into human longevity is thus making considerable progress.

Human longevity is in part genetically determined, and the insulin/IGF-1 signal transduction (IIS) pathway has consistently been implicated. In humans, type 2 diabetes is a frequent disease that results from loss of glucose homeostasis and for which new candidate polymorphisms now rapidly emerge from genome wide association studies. In the Leiden Longevity Study (n=2415), the offspring of long lived siblings ("offspring") who are genetically enriched for longevity were shown to have a more beneficial metabolic profile compared to their environmentally matched partners ("controls"), including better glucose tolerance. We tested whether the "offspring" carry a lower burden of diabetes risk alleles. Fifteen polymorphisms derived from genome wide association (GWA) scans in type 2 diabetes were tested for association with parameters of glucose metabolism in offspring and controls, and burden of risk alleles was compared between offspring and controls. Among all participants, a higher number of type 2 diabetes risk alleles associated with a higher prevalence of diabetes (P=0.011) and higher serum concentration of glucose (P<0.016) but not insulin (P=0.450). None of the polymorphisms differed in frequency between the offspring and controls (all P>0.05), nor did the mean total number of risk alleles (P=0.977). The association between polymorphisms and glucose levels did not differ between controls and offspring (Pinteraction=0.523). The better glucose tolerance of the "offspring" is not explained by a lower burden of type 2 diabetes risk alleles, suggesting that specific mechanisms determining longevity exist.

Context: A relation between low thyroid activity and prolonged life span in humans has been observed. Several studies have demonstrated hereditary and genetic influences on thyroid function., Objective: The objective of the study was to test whether low thyroid activity associated with extreme longevity constitutes a heritable phenotype, which could contribute to the familial longevity observed in the Leiden Longevity Study., Design: This was a cross-sectional study., Setting: The study was conducted at a university hospital in the city of Leiden, The Netherlands., Participants: Eight hundred fifty-nine nonagenarian siblings (median age 92.9 yr) from 421 long-lived families participated in the study. Families were recruited from the entire Dutch population if at least two long-lived siblings were alive and fulfilled the age criterion of age of 89 yr or older for males and 91 yr or older for females. There were no selection criteria on health or demographic characteristics., Intervention: Blood samples were taken for determination of serum parameters of thyroid function., Main Outcome Measure: We calculated the family mortality history score of the parents of the nonagenarian siblings and related this to thyroid function parameters in the nonagenarian siblings., Results: We found that a lower family mortality history score (less mortality) of the parents of nonagenarian siblings was associated with higher serum TSH levels (P = 0.005) and lower free T(4) levels (P = 0.002) as well as lower free T(3) levels (P = 0.034) in the nonagenarian siblings., Conclusions: Our findings support the previous observation that low thyroid activity in humans constitutes a heritable phenotype that contributes to exceptional familial longevity observed in the Leiden Longevity Study.

Background: The hypothalamo-pituitary-thyroid axis has been widely implicated in modulating the aging process. Life extension effects associated with low thyroid hormone levels have been reported in multiple animal models. In human populations, an association was observed between low thyroid function and longevity at old age, but the beneficial effects of low thyroid hormone metabolism at middle age remain elusive., Methods: We have compared serum thyroid hormone function parameters in a group of middle-aged offspring of long-living nonagenarian siblings and a control group of their partners, all participants of the Leiden Longevity Study., Results: When compared with their partners, the group of offspring of nonagenarian siblings showed a trend toward higher serum thyrotropin levels (1.65 vs157 mU/L, p = .11) in conjunction with lower free thyroxine levels (15.0 vs 15.2 pmol/L, p = .045) and lower free triiodothyronine levels (4.08 vs 4.14 pmol/L, p = .024)., Conclusions: Compared with their partners, the group of offspring of nonagenarian siblings show a lower thyroidal sensitivity to thyrotropin. These findings suggest that the favorable role of low thyroid hormone metabolism on health and longevity in model organism is applicable to humans as well.

Objectives: To explore measures of metabolic syndrome and glucose metabolism in families with exceptional longevity., Design: Case-control study., Setting: A university hospital in Leiden, the Netherlands., Participants: One hundred twenty-one offspring of nonagenarian siblings, who were enriched for familial factors promoting longevity, and 113 of their partners. No subject had diabetes mellitus., Measurements: Prevalence of metabolic syndrome was determined according to the criteria of the Third Report of the National Cholesterol Education Program. Glucose tolerance was assessed according to a 2-hour oral glucose tolerance test., Results: The offspring of nonagenarians siblings had a lower prevalence of metabolic syndrome (P=.03), similar body composition, lower mean fasting blood glucose levels (4.99 vs 5.16 mmol/L; P=.01), lower mean fasting insulin levels (5.81 vs 6.75 mU/L; P=.04), a higher mean homeostasis model assessment of insulin sensitivity (0.78 vs 0.65; P=.02), and a more-favorable glucose tolerance (mean area under the receiver operating characteristic curve for glucose (13.2 vs 14.3; P=.007) than their partners. No significant differences were observed between the offspring and their partners in beta-cell function (insulogenic index 13.6 vs 12.5; P=.38)., Conclusion: Despite similar body composition, the offspring of nonagenarian siblings showed a lower prevalence of metabolic syndrome and better glucose tolerance than their partners, centralizing the role of favorable glucose metabolism in familial longevity.

Objectives: To compare the risk of mortality of nonagenarian siblings with that of sporadic nonagenarians (not selected on having a nonagenarian sibling) and to compare the prevalence of morbidity in their offspring with that of the offsprings' partners., Design: Longitudinal (mortality risk) and cross-sectional (disease prevalence)., Setting: Nationwide sample., Participants: The Leiden Longevity Study consists of 991 nonagenarian siblings derived from 420 Caucasian families, 1,365 of their offspring, and 621 of the offsprings' partners. In the Leiden 85-plus Study, 599 subjects aged 85 were included, of whom 275 attained the age of 90 (sporadic nonagenarians)., Measurements: All nonagenarian siblings and sporadic nonagenarians were followed for mortality (with a mean+/-standard deviation follow-up time of 2.7+/-1.4 years and 3.0+/-1.5 years, respectively). Information on medical history and medication use was collected for offspring and their partners., Results: Nonagenarian siblings had a 41% lower risk of mortality (P<.001) than sporadic nonagenarians. The offspring of nonagenarian siblings had a lower prevalence of myocardial infarction (2.4% vs 4.1%, P=.03), hypertension (23.0% vs 27.5%, P=.01), diabetes mellitus (4.4% vs 7.6%, P=.004), and use of cardiovascular medication (23.0% vs 28.9%, P=.003) than their partners., Conclusion: The lower mortality rate of nonagenarian siblings and lower prevalence of morbidity in their middle-aged offspring reinforce the notion that resilience against disease and death have similar underlying biology that is determined by genetic or familial factors.

Recently, we have shown that compared to controls, long-lived familial nonagenarians (mean age: 93.4 years) from the Leiden Longevity Study displayed a lower mortality rate, and their middle-aged offspring displayed a lower prevalence of cardio-metabolic diseases, including diabetes mellitus. The evolutionarily conserved insulin/IGF-1 signaling (IIS) pathway has been implicated in longevity in model organisms, but its relevance for human longevity has generated much controversy. Here, we show that compared to their partners, the offspring of familial nonagenarians displayed similar non-fasted serum levels of IGF-1, IGFBP3 and insulin but lower non-fasted serum levels of glucose, indicating that familial longevity is associated with differences in insulin sensitivity.

Ample evidence from model organisms has indicated that subtle variation in genes can dramatically influence lifespan. The key genes and molecular pathways that have been identified so far encode for metabolism, maintenance and repair mechanisms that minimize age-related accumulation of permanent damage. Here, we describe the evolutionary conserved genes that are involved in lifespan regulation of model organisms and humans, and explore the reasons of discrepancies that exist between the results found in the various species. In general, the accumulated data have revealed that when moving up the evolutionary ladder, together with an increase of genome complexity, the impact of candidate genes on lifespan becomes smaller. The presence of genetic networks makes it more likely to expect impact of variation in several interacting genes to affect lifespan in humans. Extrapolation of findings from experimental models to humans is further complicated as phenotypes are critically dependent on the setting in which genes are expressed, while laboratory conditions and modern environments are markedly dissimilar. Finally, currently used methodologies may have only little power and validity to reveal genetic variation in the population. In conclusion, although the study of model organisms has revealed potential candidate genetic mechanisms determining aging and lifespan, to what extent they explain variation in human populations is still uncertain.

In the nematode Caenorhabditis elegans, nuclear hormone receptor DAF-12 regulates the decision to go into a resistant dauer diapause, in which the worm exhibits a decreased rate of aging. Using sequence similarity searches, we previously identified the liver X receptor alpha (LXRalpha) as one of the human nuclear hormone receptors the protein sequence of which is most similar to C. elegans DAF-12. Here, we studied whether variation in the gene encoding LXRalpha associates with human life span. In the Leiden 85-Plus Study, a population-based prospective follow-up study, we genotyped four polymorphisms spanning the gene coding for LXRalpha (NR1H3) and tagged four common haplotypes. Among 563 participants, haplotype 2 associated with reduced mortality during the 7-year follow-up (hazard ratio 0.78; p =.015), predominantly caused by reduced mortality from infectious disease (hazard ratio 0.31; p =.023). Haplotype 2 also associated with higher levels of plasma apolipoprotein E, a target gene of the LXRalpha (p =.018), and higher levels of triglycerides (p =.041). Genetic variation in the gene coding for the LXRalpha (NR1H3) associates with human life span.

Earlier studies on human fibroblast cultures have demonstrated an inverse relationship between the total number of population doublings (PDs) and donor age. As more recent studies were unable to replicate these findings, we set out to analyze growth characteristics of fibroblast cultures from nonagenarians who represent the extreme of human lifespan. Therefore, we obtained skin biopsies from 68 participants of the Leiden 85-plus Study, all aged 90 years. None of the 68 strains failed to proliferate and all were easily cultured under highly standardized conditions. Within a time window of 30 months, all strains displayed a high and reproducible replicative capacity that was maintained for at least 50 PDs. A decline in mitotic activity was observed between 26 and 81 PDs. Out of the 68 cell strains, 58 strains reached the post-mitotic phase with an onset between 51 and 108 PDs. The growth pattern of each senescent strain was fitted by a piecewise linear model, which allowed calculation of the transition towards the phase of decreased growth speed, as well as by a nonlinear continuous model; goodness-of-fit was high and not different between the models (both > 0.99). Growth characteristics were not associated with morbidity or mortality of the donors. We conclude that fibroblasts from nonagenarians maintain a high-replicative capacity despite a huge variability in the onset of senescence. These results cast further doubt on the association between in vitro growth characteristics of fibroblast cultures and the length of human lifespan.

Evidence is accumulating that aging is hormonally regulated by an evolutionarily conserved insulin/IGF-1 signalling (IIS) pathway. Mutations in IIS components affect lifespan in Caenorhabditis elegans, Drosophila melanogaster and mice. Most long-lived IIS mutants also show increased resistance to oxidative stress. In D. melanogaster and mice, the long-lived phenotype of several IIS mutants is restricted to females. Here, we analysed the relationship between IIS signalling, body height and longevity in humans in a prospective follow-up study. Based on the expected effects (increased or decreased signalling) of the selected variants in IIS pathway components (GHRHR, GH1, IGF1, INS, IRS1), we calculated composite IIS scores to estimate IIS pathway activity. In addition, we analysed the relative impact on lifespan and body size of the separate variants in multivariate models. In women, lower IIS scores are significantly associated with lower body height and improved old age survival. Multivariate analyses showed that these results were most pronounced for the GH1 SNP, IGF1 CA repeat and IRS1 SNP. In females, for variant allele carriers of the GH1 SNP, body height was 2 cm lower (P = 0.007) and mortality 0.80-fold reduced (P = 0.019) when compared with wild-type allele carriers. Thus, in females, genetic variation causing reduced IIS activation is beneficial for old age survival. This effect was stronger for the GH1 SNP than for variation in the conserved IIS genes that were found to affect longevity in model organisms.

Longevity may depend on a balance between tumor suppression and tissue renewal mechanisms [Campisi, J., 2003. Cancer and ageing: rival demons? Nat. Rev. Cancer 3 (5), 339-349]. Mice with constitutively activated p53 are almost cancer free but their life span is reduced and accompanied by early tissue atrophy [Tyner et al., 2002. p53 mutant mice that display early ageing-associated phenotypes. Nature 415 (6867) 45-53]. Replacement of arginine (Arg) by proline (Pro) at position 72 of human p53 decreases its apoptotic potential [Dumont et al., 2003. The codon 72 polymorphic variants of p53 have markedly different apoptotic potential. Nat. Genet. 33 (3), 357-365] providing a tool to test for a similar trade-off in humans. Using a formal meta-analysis of the published literature we show that carriers of the TP53 codon 72 Pro/Pro genotype have an increased cancer risk compared to Arg/Arg carriers (p<0.05). Next, in a prospective study of 1226 people aged 85 years and over we show that carriers of the Pro/Pro genotype have a 41% increased survival (p = 0.032) despite a 2.54 fold increased (p = 0.007) proportional mortality from cancer. It is suggested that human p53 protect against cancer but at a cost of longevity.

Mice in which the p66(SHC) specific region of the SHC gene is deleted live 30% longer without apparent disease. These mice have lower levels of oxidative stress and apoptosis, both of which have been linked to old age survival in man. This makes SHC1 an important candidate gene for longevity in humans. We found no variations in the p66 specific region of the SHC1 gene in 30 young and 30 extreme long-lived subjects. Thus in man, no common sequence variations occur in p66 specific region of the SHC1 gene. In two independent cohorts of respectively 730 and 563 subjects aged 85 and over, we tested the only known non-synonymous polymorphism, Met(410)Val, for association with longevity using a prospective follow-up design. In the first cohort, we found increasing valine allele frequency in three strata of increasing age at death (2.8-5.2%). Moreover, compared to Met/Met carriers, mortality rate was a factor of 0.71 (95% CI 0.45-1.13) reduced for Met/Val carriers in the combined cohorts, with similar risk estimates in both cohorts. Low valine allele frequency resulted, however, in low power to detect statistical significance. These data suggest that an association between the Met(410)Val polymorphism and longevity in humans may exist.