Bioactive growth hormone in older men and women: It's relationship to immune markers and healthspan.

OBJECTIVE: The consequences of age-related decline in the somatotropic axis of humans are complex and remain largely unresolved. We tested the hypothesis that hGH measurements of plasma by bioassay vs immunoassay from samples obtained from free-living, elderly individuals would reveal a dichotomy in GH activities that are correlated with the functional status of the donors, i.e. their healthspan. DESIGN: Forty-one men and women of advanced age (men: N=16, age, 80.5±6.5years; height, 173.1±6.9cm; body mass, 81.8±13.0kg) and (women: N=25, age, 80.7±7.2years; height, 157.7±6.0cm; body mass, 68.8±17kg), were recruited for a cross-sectional study. Participants filled out PROMIS (Patient-Reported Outcomes Measurement Information System, U. S. Department of Health and Human Services) scales, undertook physical performance tests and had fasted blood samples obtained at rest for measurement of hormonal and immunology biomarkers. RESULTS: When measured by the well-established rat tibial line GH bioassay, one half of the plasma samples (n=20) contained bioassayable GH (bGH), but the other half (n=21) failed to mount increases in tibial plate width above saline injected controls. This difference did not correlate with the age, sex or physical functionality of the plasma donor. It also did not correlate with hGH concentrations measured by immunoassay. In those cases in which bGH was detected, various hierarchical regression models predicted that GHRH, c-peptide, VEGF, NPY, IL-4 and T-regulatory lymphocytes were associated with the difference and predicted bGH. CONCLUSION: Results from this study suggest that the actions of bGH at the cellular level may be modified by other factors and that this may explain the lack of correlations observed in this study.

Significant life extension by ten percent dietary restriction.

Although it is well documented that dietary restriction (DR) increases the life span of rodents and other animals, this increase is observed at relatively high levels of DR, in which rodents are typically fed 40% less than that consumed by rodents fed ad libitum. It is generally assumed that lower levels of DR will have a lesser impact on life span; however, there are very little published data on the effect of low levels of DR on life span. In this study, we show that 10% DR increased life span to almost the same extent as 40% DR. While both 10% and 40% DR resulted in similar changes in non-neoplastic lesions, 10% DR had no significant effect on the incidence of neoplasia (except for pituitary adenoma), and 40% DR resulted in a significant reduction (40%) in neoplasia. These data clearly demonstrate that the life span of F344 rats does not increase linearly with the level of DR; rather, even a low level of DR can substantially affect life span. This rodent study has important translational implications because it suggests that a modest reduction in calories might have significant health benefits for humans.

Altered metabolism and resistance to obesity in long-lived mice producing reduced levels of IGF-I.

The extension of lifespan due to reduced insulin-like growth factor 1 (IGF-I) signaling in mice has been proposed to be mediated through alterations in metabolism. Previously, we showed that mice homozygous for an insertion in the Igf1 allele have reduced levels of IGF-I, are smaller, and have an extension of maximum lifespan. Here, we tested whether this specific reduction of IGF-I alters glucose metabolism both on normal rodent chow and in response to high-fat feeding. We found that female IGF-I-deficient mice were lean on a standard rodent diet but paradoxically displayed an insulin-resistant phenotype. However, these mice gained significantly less weight than normal controls when placed on a high-fat diet. In control animals, insulin response was significantly impaired by high-fat feeding, whereas IGF-I-deficient mice showed a much smaller shift in insulin response after high-fat feeding. Gluconeogenesis was also elevated in the IGF-I-deficient mice relative to controls on both normal and high-fat diet. An analysis of metabolism and respiratory quotient over 24 h indicated that the IGF-I-deficient mice preferentially utilized fatty acids as an energy source when placed on a high-fat diet. These results indicate that reduction in the circulating and tissue IGF-I levels can produce a metabolic phenotype in female mice that increases peripheral insulin resistance but renders animals resistant to the deleterious effects of high-fat feeding.

Mice producing reduced levels of insulin-like growth factor type 1 display an increase in maximum, but not mean, life span.

Reduced signaling through the IGF type 1 (IGF-1) receptor increases life span in multiple invertebrate organisms. Studies on mammalian longevity suggest that reducing levels of IGF-1 may also increase life span. However, the data are conflicting and complicated by the physiology of the mammalian neuroendocrine system. We have performed life-span analysis on mice homozygous for an insertion in the Igf1 gene. These mice produce reduced levels of IGF-1 and display a phenotype consistent with a significant decrease in IGF-1. Life-span analysis was carried out at three independent locations. Although the life-span data varied between sites, the maximum life span of the IGF-1-deficient mice was significantly increased and age-specific mortality rates were reduced in the IGF-1-deficient mice; however, mean life span did not differ except at one site, where mean life span was increased in female IGF-1-deficient animals. Early life mortality was noted in one cohort of IGF-1-deficient mice. The results are consistent with a significant role for IGF-1 in the modulation of life span but contrast with the published life-span data for the hypopituitary Ames and Snell dwarf mice and growth hormone receptor null mice, indicating that a reduction in IGF-1 alone is insufficient to increase both mean and maximal life span in mice.

Frailty consensus: a call to action.

Frailty is a clinical state in which there is an increase in an individual's vulnerability for developing increased dependency and/or mortality when exposed to a stressor. Frailty can occur as the result of a range of diseases and medical conditions. A consensus group consisting of delegates from 6 major international, European, and US societies created 4 major consensus points on a specific form of frailty: physical frailty. 1. Physical frailty is an important medical syndrome. The group defined physical frailty as "a medical syndrome with multiple causes and contributors that is characterized by diminished strength, endurance, and reduced physiologic function that increases an individual's vulnerability for developing increased dependency and/or death." 2. Physical frailty can potentially be prevented or treated with specific modalities, such as exercise, protein-calorie supplementation, vitamin D, and reduction of polypharmacy. 3. Simple, rapid screening tests have been developed and validated, such as the simple FRAIL scale, to allow physicians to objectively recognize frail persons. 4. For the purposes of optimally managing individuals with physical frailty, all persons older than 70 years and all individuals with significant weight loss (>5%) due to chronic disease should be screened for frailty.

Association of ghrelin receptor promoter polymorphisms with weight loss following Roux-en-Y gastric bypass surgery.

BACKGROUND: Ghrelin plays a role in appetite and has been hypothesized to play a role in the mechanism of Roux-en-Y gastric bypass (RYGB) surgery. Single nucleotide polymorphisms (SNPs) in the promoter region of its receptor gene (growth hormone secretagogue receptor type 1a--GHSR) have also been associated with weight loss outcomes following long-term dietary intervention in adults with impaired glucose tolerance. Our objectives were to evaluate changes in serum ghrelin levels and determine the effect of GHSR promoter polymorphisms on post-RYGB surgery weight loss. METHODS: Preoperative and 6-month postoperative serum ghrelin levels were measured in 37 patients with extreme obesity undergoing RYGB surgery. Total ghrelin was also measured in liver tissue collected intraoperatively. Association analysis between genotypes for SNPs rs9819506 and rs490683 in the promoter region of the GHSR gene and weight loss outcomes in the 30 months following surgery was performed in over 650 RYGB patients. RESULTS: Serum ghrelin levels increased after RYGB surgery. Weight loss trajectories were significantly different using an additive model for both ghrelin SNPs, with patients homozygous for the rs490683 CC genotype exhibiting the most weight loss. Weight loss trajectories were also different using a dominant model. The rs490683 risk allele demonstrated decreased promoter activity in vitro. CONCLUSIONS: The role of increased ghrelin levels in weight loss outcomes following RYGB surgery may be influenced by variation in the GHSR gene.

Heterogeneous stocks and selective breeding in aging research.

There are advantages and limitations to using genetically heterogeneous stocks and selective breeding procedures in gerontological and health span research. Animal models that address complex systems of aging involve constraint or manipulation of genetic diversity. They derive from levels of genetic analysis ranging from molecular to quantitative and are relevant to levels of causal hierarchy from base sequences to complex multivariate phenotypes. For some research purposes control of the genotypic source of phenotypic variability by fixation is desirable; others involve establishing genetic diversity or deliberately manipulating identified genes or anonymous gene complexes to specification. Genetic heterogeneity is essential or advantageous for multivariate description of complex phenomena, the examination of associations among variables, or manipulation of polygenic systems. This review concentrates on these latter quantitative requirements. Space limitations preclude a comprehensive review, but relevant sample references and some hints of historical perspectives are provided, with apologies to the many relevant authors not cited.

Long-term IGF-I exposure decreases autophagy and cell viability.

A reduction in IGF-I signaling has been found to increase lifespan in multiple organisms despite the fact that IGF-I is a trophic factor for many cell types and has been found to have protective effects against multiple forms of damage in acute settings. The increase in longevity seen in response to reduced IGF-I signaling suggests that there may be differences between the acute and chronic impact of IGF-I signaling. We have examined the possibility that long-term stimulation with IGF-I may have a negative impact at the cellular level using quiescent human fibroblasts. We find that fibroblast cells exposed to IGF-I for 14 days have reduced long-term viability as judged by colony forming assays, which is accompanied by an accumulation of senescent cells. In addition we observe an accumulation of cells with depolarized mitochondria and a reduction in autophagy in the long-term IGF-I treated cultures. An examination of mice with reduced IGF-I levels reveals evidence of enhanced autophagy and fibroblast cells derived from these mice have a larger mitochondrial mass relative to controls indicating that changes in mitochondrial turnover occurs in animals with reduced IGF-I. The results indicate that chronic IGF-I stimulation leads to mitochondrial dysfunction and reduced cell viability.

The biological sciences section program at the 60th Annual Meeting of the Gerontological Society of America.

In this era of genomics and other exciting technical advances, research on the biology of aging is undergoing a renaissance. This report summarizes 10 cutting-edge areas of research covered in symposia that spanned such topics as stem cells, novel vaccine strategies, nutritional sensing, new concepts of Parkinson's disease, high throughput screening for aging interventions, manipulating telomerase in cancer and immunodeficiency, synergy between aging and HIV disease, and epigenetic influences on aging. Novel animal models, including those showing no evidence of aging, as well as ethical and political implications of embryonic stem cells and alternative medicine are also discussed.

Plasma glucose and the action of calorie restriction on aging.

We tested the hypothesis that retardation of aging by caloric restriction is due in part to decreased levels of plasma glucose over the life span. Male C57BL/6 mice expressing a human GLUT4 minigene (transgenic [TG] mice) and their nontransgenic littermates (NTG mice) were maintained under specific pathogen-free conditions. Mice were fed ad libitum (A mice) or 40% less than ad libitum (R mice) from age 6 weeks. Over the life span there were three different levels of plasma glucose, with NTGA mice having the highest daily levels, TGR mice the lowest daily values, and TGA and NTGR mice having similar levels intermediate between these values. Despite differences in plasma glucose, the differences measured in longevity (50% and 10% survival), physiology and tissue pathology were associated with diet rather than with levels of plasma glucose. We conclude that decreased plasma glucose over the life span is not an important factor in the action of calorie restriction on aging processes.

Tissue specific and non-specific changes in gene expression by aging and by early stage CR.

Aging alters the expression of a variety of genes. Calorie restriction (CR), which extends life span in laboratory rodents, also changes gene expression. This study investigated changes in gene expression across three different tissues from the same mouse to examine how aging and early stage CR influence gene expression in different tissues of an organism. Expression profiling of heart, liver, and hypothalamus tissues was done in young (4-6 months) ad libitum fed (AL), young CR (2.5-4.5 months of CR), and old (26-28 months) AL male C57BL/6 mice. Aging significantly altered the expressions of 309, 1819, and 1085 genes in heart, liver, and hypothalamus tissues, respectively. In nine genes, aging altered expression across all three tissues although the regulation directions did not agree across all three tissues for some genes. Early stage CR in young mice significantly changed the expressions of 192, 839, and 100 genes in heart, liver, and hypothalamus tissues, respectively, and seven genes altered expression across all three tissues; three were up regulated and four were down regulated. The results of Gene Ontology (GO) Biological Process analysis indicated up regulation of antigen processing/presentation genes by aging and down regulation of stress response genes by early stage CR in all three tissues. The comparison of the results of aging and short term CR studies showed there were 389 genes, 18 GO biological processes, and 20 GO molecular functions in common.

Conjugated linoleic acid and chromium lower body weight and visceral fat mass in high-fat-diet-fed mice.

More than half of the U.S. population has a body mass index of 25 kg/m2 or more, which classifies them as overweight or obese. Obesity is often associated with comorbidities such as diabetes, cardiovascular diseases, and cancer. CLA and chromium have emerged as major dietary supplements that reduce body weight and fat mass, and increase basal metabolic rate in animal models. However, studies show that CLA induces insulin resistance in mice and in humans, whereas Cr improves insulin sensitivity. Hence, we designed the present study to examine the combined effect of CLA and Cr on body composition and insulin sensitivity in a Balb/c mice (n = 10/group) model of high-fat-diet-induced obesity. CLA alone lowered body weight, total body fat mass, and visceral fat mass, the last of which decreased further with the combination of CLA and Cr. This effect was accompanied by decreased serum leptin levels in CLA-fed and CLA + Cr-fed mice, and by higher energy expenditure (EE) and oxygen consumption (OC) in CLA + Cr-fed mice. Serum levels of glucose, insulin, the pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6), as well as insulin resistance index (IRI), decreased with CLA, whereas CLA and Cr in combination had significant effects on insulin and IL-6 concentrations and IRI. In summary, CLA + Cr decreased body weight and fat mass in high-fat-diet-fed mice, which may be associated with decreased leptin levels and higher EE and OC.

Early hypothalamic response to age-dependent gene expression by calorie restriction.

Molecular events linking the initial detection of calorie restriction (CR) to changes in gene expression throughout the organism that ultimately retard aging in CR animals are unknown. This study measured changes in gene expression induced by CR and by aging in the hypothalamus, which likely plays a central role in the initial perception of and response to CR. Hypothalamic expression profiling was done in young (4-6 months) ad libitum fed (AL), young CR (2.5-4.5 months of CR), and old (26-28 months) AL male C57BL/6 mice. CR altered the expression of 137 genes and aging altered 1222. Only 8 age-related genes were oppositely regulated by CR. To test whether reduced plasma glucose is a signal in altering hypothalamic gene expression, we examined GLUT4 transgenic mice (C57BL/6 background; 4-6 months), which have reduced plasma glucose similar to that of CR mice. Twenty-seven genes differed between transgenic and non-transgenic mice; nine of these were only altered by CR. The decreased plasma glucose had a limited role in CR mediated hypothalamic gene expression.

The combination of dietary conjugated linoleic acid and treadmill exercise lowers gain in body fat mass and enhances lean body mass in high fat-fed male Balb/C mice.

Nearly half of the U.S. adult population is overweight or obese, which may be related to increased energy intake combined with lack of physical activity. Obesity increases the risk of several chronic diseases including diabetes, coronary heart disease, hypertension, and stroke. Conjugated linoleic acids (CLA) were shown to decrease fat and increase lean mass in several animal studies. However, the effects of CLA in combination with exercise (Ex) on body composition have not been studied in an animal model. We examined the effect of a low concentration of either safflower oil as control (0.5%) or mixed isomers of CLA (0.4%) along with treadmill exercise on body composition in male Balb/C mice fed a high-fat diet (20% corn oil) in a 2 x 2 factorial design. CLA consumption lowered change in fat mass (P < 0.001) confirming the results of other studies, and change in fat mass decreased further (P < 0.001) with CLA and exercise. Change in lean mass did not increase with exercise alone; it increased, although not significantly, with CLA alone and increased significantly (P < 0.05) due to the combination of CLA and exercise. This effect was accompanied by decreased serum leptin levels and lower leptin mRNA expression in peritoneal fat (P < 0.001). Serum insulin, glucose, tumor necrosis factor (TNF)-alpha, and interleukin-6 were lower in CLA-fed mice than in controls (P < 0.05), whereas serum TNF-alpha was increased by exercise (P < 0.05). Exercise increased oxygen consumption and energy expenditure when measured under resting conditions (P < 0.05). In summary, the combination of dietary CLA and exercise decreased fat mass and increased lean mass in mice fed a high-fat diet, and these effects may be related in part to decreased serum leptin and exercise-induced increases in oxygen consumption and energy expenditure.

Hepatic genes altered in expression by food restriction are not influenced by the low plasma glucose level in young male GLUT4 transgenic mice.

Because food restriction (FR) has a profound effect on most tissues, it is plausible that the modulation of aging by FR occurs through cellular processes such as gene expression. The effect of FR in lowering plasma glucose levels has been demonstrated in mice, rats, and nonhuman primates. The consistency of this finding suggests that decreased plasma glucose may be an important consequence of FR. Indeed, lowering plasma glucose in the absence of FR would be expected to change the expression of some of the same genes as seen with FR. GLUT4 transgenic (TG) mice were particularly suited to this examination because they have low plasma glucose levels like FR mice. We investigated altered gene expression by FR and the effect of low plasma glucose levels caused by genetic manipulation by measuring mRNA expression in liver tissues of 4- to 6-mo-old mice with 2.5-4.5 mo of FR using microarrays and 4 groups: GLUT4 TG (C57BL/6 background) consumed food ad libitum (AL), GLUT4 TG FR, wild-type littermates AL, and wild-type littermates FR. The 3 statistical analysis methods commonly indicated that FR altered the expression of 1277 genes; however, none of these genes was altered by additional GLUT4 expression. In fact, the low plasma glucose level in GLUT4 TG mice did not affect gene expression. Some results were confirmed by real-time quantitative RT-PCR. We conclude that a low plasma glucose level does not contribute to or coincide with the effect of FR on gene expression in the liver.

Reproducibility, sources of variability, pooling, and sample size: important considerations for the design of high-density oligonucleotide array experiments.

We have undertaken a series of experiments to examine several issues that directly affect design of gene expression studies using Affymetrix GeneChip arrays: probe-level analysis, need for technical replication, relative contribution of various sources of variability, and utility of pooling RNA from different samples. Probe-level data were analyzed by Affymetrix MAS 5.0, and three model-based methods, PM-MM and PM-only models by dChip, and the RMA model by Bioconductor, with the latter two providing the best performance. We found that replicate chips of the same RNA have limited value in reducing total variability, and for relatively highly expressed genes in this biologically homogeneous animal model of aging, about 11% of total variation is due to day effects and the remainder is approximately equally split between sample and residual sources. We also found that pooling samples is neither advantageous nor detrimental. Finally we suggest a strategy for sample size calculations using formulas appropriate when coefficients of variation are known, target effects are expressed as fold changes, and data can be assumed to be approximately lognormally distributed.

Time to talk SENS: critiquing the immutability of human aging.

Aging is a three-stage process: metabolism, damage, and pathology. The biochemical processes that sustain life generate toxins as an intrinsic side effect. These toxins cause damage, of which a small proportion cannot be removed by any endogenous repair process and thus accumulates. This accumulating damage ultimately drives age-related degeneration. Interventions can be designed at all three stages. However, intervention in metabolism can only modestly postpone pathology, because production of toxins is so intrinsic a property of metabolic processes that greatly reducing that production would entail fundamental redesign of those processes. Similarly, intervention in pathology is a "losing battle" if the damage that drives it is accumulating unabated. By contrast, intervention to remove the accumulating damage would sever the link between metabolism and pathology, and so has the potential to postpone aging indefinitely. We survey the major categories of such damage and the ways in which, with current or foreseeable biotechnology, they could be reversed. Such ways exist in all cases, implying that indefinite postponement of aging--which we term "engineered negligible senescence"--may be within sight. Given the major demographic consequences if it came about, this possibility merits urgent debate.