Roy L. Walford

Calorie restriction inhibits the age-related dysregulation of the cytokines TNF-α and IL-6 in C3B10RF1 mice

TNF-alpha and IL-6 are generally increased in the sera of aged humans and mice. The dysregulation of these cytokines may be critical in autoreactivity and immune dysfunction. In earlier studies we demonstrated that production of TNF-alpha and IL-6 following in vitro stimulation of peritoneal macrophages by LPS was reduced in old compared to young mice, and that dietary caloric restriction (CR) had no effect on the induction of TNF-alpha in this system. In the present study we examined the effects of age and calorie restriction on the constitutive production of both TNF-alpha and IL-6. Serum levels of both cytokines were significantly higher in old versus young mice. However, in old mice subjected to long term CR the serum levels were comparable to those of young mice. The potential involvement of normalization of TNF-alpha and IL-6 levels in the life extension effect of CR are discussed.

The retardation of aging by caloric restriction : its significance in the transgenic era

The retardation of aging and diseases by caloric restriction (CR) is a widely-studied and robust phenomenon. Recent publications describe transgenic and other mutant rodents displaying lifespan extension, and the rapid pace at which these animals are being generated raises the possibility that the importance of the CR paradigm is declining. Here we discuss these models and evaluate the evidence whether or not the aging process is retarded based on longevity, disease patterns and age-associated biological changes. A comparison to rodents on CR is made. Because CR has been investigated for approximately 70 years with increasing intensity, there exists extensive data to document aging retardation. In contrast, for nearly all of the genetically abnormal models of lifespan extension, such data are minimal and often unconvincing; additional studies will be required to validate these strains as suitable models for aging research.

Calories and aging alter gene expression for gluconeogenic, glycolytic, and nitrogen-metabolizing enzymes

We characterized the effects of calorie restriction (CR) on the expression of key glycolytic, gluconeogenic, and nitrogen-metabolizing enzymes in mice. Of the gluconeogenic enzymes investigated, liver glucose-6-phosphatase mRNA increased 1.7- and 2. 3-fold in young and old CR mice. Phosphoenolpyruvate carboxykinase mRNA and activity increased 2.5- and 1.7-fold in old CR mice. Of the key glycolytic enzymes, pyruvate kinase mRNA and activity decreased approximately 60% in CR mice. Hepatic phosphofructokinase-1 and pyruvate dehydrogenase mRNA decreased 10-20% in CR mice. Of the genes that detoxify ammonia generated from protein catabolism, hepatic glutaminase, carbamyl phosphate synthase I, and tyrosine aminotransferase mRNAs increased 2.4-, 1.8-, and 1.8-fold with CR, respectively. Muscle glutamine synthetase mRNA increased 1.3- and 2. 1-fold in young and old CR mice. Hepatic glutamine synthetase mRNA and activity each decreased 38% in CR mice. These CR-induced changes are consistent with other studies suggesting that CR may decrease enzymatic capacity for glycolysis and increase the enzymatic capacity for hepatic gluconeogenesis and the disposal of byproducts of muscle protein catabolism.

Modification of mitochondrial respiration by aging and dietary restriction.

Effects of aging and of dietary restriction on mitochondrial recovery and respiratory capacities have been assessed in mice. Old mice (23-26 months) did not differ from adult mice (9-12 months) in amounts of protein recovered in mitochondrial fractions of liver, brain and spleen, but did show a decline in specific activity of cytochrome c oxidase (cyt. c ox.) in liver and spleen. Age effects on in vitro respiration by mitochondria occurred in liver and spleen. In liver, only one substrate (beta-hydroxybutyrate) of four tested was respired at a different rate by old than by young mitochondria. Depression of state 3 respiration and 2,4-dinitrophenol (DNP)-uncoupled rates was observed for this substrate; however, this effect depended on expressing respiration on the basis of mitochondrial protein and was less overt if data were expressed per unit of cyt. c ox. activity. Old spleen mitochondria exhibited a grosser defect, showing a 40% decrease in the respiratory control index (RCI) for (succinate + rotenone)- supported respiration (the only substrate tested) due to a possible increase in state 4 rates. Effects of dietary restriction were assessed in liver and brain of 3-7-month-old mice underfed since weaning. Dietary restriction reduced recovery of total liver mitochondrial protein and liver cyt. c ox. specific activity. Liver mitochondria from restricted mice generally showed increased state 3 rates with no differences from controls in state 4 rates for respiration supported by glutamate or pyruvate + malate, resulting in an increased RCI for these substrates. DNP-uncoupled rates were also raised by dietary restriction. Unlike effects observed in old versus young mice, these differences obtained whether the data were expressed on the basis of mitochondrial protein or on cyt. c ox. activity. Electron microscopy of liver mitochondrial preparations revealed more non-mitochondrial contaminants in old mice and larger mitochondria in dietarily restricted mice. These findings are compatible with reports of age-dependent losses of liver mitochondria and suggest that dietary restriction may retard this loss.

Human T lymphocytes possess a limited in vitro life span

The T lymphocyte offers certain theoretical advantages over other available cell types for the study of aging. Immunosenescence is a well-established part of, and may be directly relevant to, mammalian aging, and the T lymphocyte is well-characterized as to function, cell-surface antigen make-up, and other factors. However, prior efforts at studying in vitro aging of T cells have been hampered by poor reproducibility in doubling potential and the occurrence of a peculiar type of crisis. We have improved the culture conditions for long-term in vitro propagation of normal human T lymphocytes so that previously described variability between identically manipulated cultures and the crisis period have been eliminated. Analysis of the growth patterns of 109 individual cultures revealed a limited proliferative life span, with the number of cumulative population doublings corresponding to that reported for adult human fibroblasts. This accord between the in vitro life spans of two vastly different cell types lends further support to the concept of the Hayflick Limit as a general biological phenomenon.

Longterm dietary restriction and immune function in mice, response to sheep red blood cells and to mitogenic agents.

Abstract The humoral immune response to injection of sheep red blood cells and the responses of splenic lymphocytes to stimulation by three mitogens (phytohemagglutinin, concanavalin-A, and pokeweed) were compared between mice on a daily 21.6% casein/14.5 calorie diet and a population on half that amount from time of weaning. The mice were tested at 18–20 and 52–55 weeks of age. The results suggested that the more restrictive diet caused significant delay in maturation of the immune system. It is thus shown that a McCay type of dietary regime exerts a profound effect on the immune system. Whether this type of effect might be primary in the de-acceleration of aging classically associated with appropriate restriction of diet during childhood, or whether the immune changes are only tangentially related thereto is not yet established.

Influence of the main histocompatibility complex on ageing in mice.

CONSIDERATION of the facts of mammalian cell ‘transformation’ by viruses led us to the proposition that only a few genes or gene systems need necessarily be involved in the ageing process1,2. These views accord in principle with conclusions derived from more recent analyses of the genetic basis for the increase in lifespan of hominid species in the last several hundred thousand years, namely that mutations at no more than about 0.6% of the total genome could be responsible for the increase3,4. We describe here a study of ultimate lifespans (as reflected by tenth deciles of survivorship) in congenic mice which suggests that the main histocompatibility complex (MHC) is one of the gene systems involved in the control of ageing.

Serologic Typing of Human Lymphocytes with Immune Serum Obtained after Homografting

Serums obtained from paired humans following reciprocal fourthset skin homografts, or from a prolonged series of reciprocal intradermalinjections of leukocytes, displayed potent cytotoxic activity againstlymphocytes of certain individuals and no activity against lymphocytes of other persons. Study of one family suggested that lymphocyte reactivity may be transmitted according to simple Mendelian genetics. Itis probable that this reactivity reflects and can be used to identifyspecific histocompatibility antigens in man.

The accumulation of non-replicative, non-functional, senescent T cells with age is avoided in calorically restricted mice by an enhancement of T cell apoptosis

Peripheral blood lymphocytes of elderly humans show an increased percentage of T cells with characteristics of replicative senescence. Similarly, the overall decrease in T cell proliferation in aged mice reflects a progressively increasing proportion of non-functional cells rather than a uniform decline in function by all cells. The improved immune function of calorically restricted (CR) animals is, paradoxically, accompanied by a relative lymphopenia. To test whether the reduction in lymphocyte number in the CR mice might reflect more efficient elimination of T cells, we measured apoptosis in young, old and CR old mice. T cell apoptosis induced by irradiation, Staurosporine, anti-CD3, and heat shock was reduced by 62, 42, 32, and 30%, respectively, in old compared with young mice. Caloric restriction normalized apoptosis in T cells from aged mice. Enhanced elimination of non-functional T cells in CR mice may be, at least in part, responsible for their improved immune functional status relative to non-CR mice of the same age.

Survival and disease patterns in C57BL/6J mice subjected to undernutrition

This study reports survival and disease patterns in a long-lived mouse strain subjected to undernutrition. Four cohorts were studied, each composed of two or more groups of mice, each normally-fed or restricted either pre- and/or postweaning. Restriction prior to weaning was effected by limiting access to the mother. animals restricted postweaning received a nutritionally complete diet, including a normal complement of vitamins and salts, but were fed only 4 portions/week vs 7 portions/week for those animals normally fed—hence the term under-nutrition to differentiate between this and malnutrition. Comparisons of disease patterns among groups revealed that the incidence of lymphoma, the most prevalent tumor, was uniformly decreased in the groups restricted postweaning, with or without preweaning restriction. In the last cohort, deaths of animals with lymphoma were shifted to later ages in the restricted groups, compared with the normally-fed controls. Whereas the lymphoma pattern was considerably modified by undernutrition, the effect on overall survival did not seem as dramatic. Gompertzian parameters for survival past 120 weeks were not statistically different, although with one exception, maximum survival and one of the Gompertzian parameters was consistently greater in groups restricted postweaning, compared with those restricted preweaning only, or not at all. Maximum survival is a parameter not unduly influenced by environmental factors such as infectious disease; consequently, this represents a meaningful effect of undernutrition. Statistically, more significant differences in tumor patterns than in survival suggests that the former are more sensitive to undernutrition than is the latter—at least in this strain of mouse. Greater lifespan prolongation in the restricted animals may be possible through better “fine tuning” of the diet, including improved portion control, particularly in the early postweaning period, to prevent rapid weight gain, and possibly through changes in dietary composition. Finally, it is suggested that undernutrition may exert its effects through an alteration in gene expression.