The serious search for an anti- aging pill. Mark Lane, Donald Ingram George Roth

Scientists found over 60 years ago that rats fed a calorie restricted (CR) diet lived longer on average than free-feeding rats and had a reduced incidence of conditions that become increasingly common in old age. Unfortunately, for maximum benefit, people would probably have to reduce their caloric intake by roughly 30 percent, equivalent to dropping from 2,500 calories a day to 1,750.

Many physiological and biochemical changes induced by caloric restriction led to delaying aging in mammals. Lane et. al believed that changes in cellular metabolism -- uptake of nutrients from the blood and their conversion to energy usable for cellular activities --would be key. Would changes related to metabolism of the sugar glucose would account for the benefits of caloric restriction?

Glucose forms when the body digests carbohydrates. It is the primary source of energy in the body--that is, it is the main material used by cells for making ATP, or adenosine triphosphate, the molecule that directly powers most cellular activities. Insulin is secreted as glucose levels in the blood rise after a meal, and it serves as the key that opens cell "doors" to the sugar.

Lane et al. concentrated on glucose and insulin because reductions in their levels and increases in cellular sensitivity to insulin are among the most consistent hallmarks of caloric restriction in both rodents and primates, occurring very soon after restriction is begun.

Lane et al searched the scientific literature for ways to manipulate insulin secretion and sensitivity without causing diabetes or its opposite, hypoglycemia. Found studies from the 1940s and 1950s on 2-deoxy-D-glucose (2DG) that reportedly lowered insulin levels in the blood

The compound apparently reproduced many classic responses to caloric restriction--among them reduced tumor growth (a response only slightly less robust than the well-known extension of life span), lowered temperature, elevated levels of glucocorticoid hormones and reduced numbers of reproductive cycles

2DG disrupts the functioning of a key enzyme involved in processing glucose in cells. The compound structurally resembles glucose, so it enters cells readily. enzyme that completes the next of several steps involved in glucose processing essentially chokes on the intermediate produced from 2DG. its ability to act on the normal glucose intermediate becomes impaired.

The net result is that cells make smaller amounts of glucose's by-products, just as occurs when caloric restriction limits the amount of glucose going into cells. Certain of these products serve as the raw material for the ATP-making machinery, which is composed of a series of protein complexes located in intracellular compartments called mitochondria. Deprived of this raw material, the machinery makes less ATP.

First experiments Delivered low 2DG doses to rats by adding it to their feed for six months. The treatment moderately reduced fasting blood glucose levels (levels measured after food was removed for 12 hours), body weight and temperature, and robustly reduced fasting insulin levels--findings consistent with the actions of caloric restriction itself.

2DG has a fatal flaw Though safe at certain low levels, it apparently becomes toxic for some animals when the amount delivered is raised just a bit or given over long periods. The narrowness of the safety zone separating helpful and toxic doses would bar it from human use.

Nutritional Control of Aging Zimmerman, Malloy, Krajcik-May

Hypothesis Life extension may be achieved with reduced amounts of the amino acids tryptophan or methionine in diet, without reduced caloric intake.

Materials 344 male rats at 4 weeks of age with 3 rats per cage Control feed (Purina Rat Chow) Methionine reduced (MR) feed

Methods Pre-fed for 2 weeks on Purina Rat Chow MR and Control fed the same quantity of food Separated into 2 groups at 6 weeks of age Fed Control or MR diet at 8 weeks of age Control diet has 0.86% methionine MR diet has of 0.17% methionine Same amount of food available to both.

Results of MR diet significantly stunted growth extended life by 42% mean and 46% maximal longevity MR ate less food than CR.

Conclusions Hypthesis is not proven. Results confounded because MR rats were smaller and ate less food MR caused a variety of metabolic and physiological changes. Some changes are like CR, some are different. Shows problem with experiment design: MR caused rats to eat less, confounded effects with CR

A C. elegans mutant that lives twice as long as wild type. Kenyon C, Chang J, Gensch E, Rudner A, Tabtiang R.

Dauer Dauer is an arrested state in young larvae analogous to hibernation or spore formation. developmentally arrested, sexually immature restricted to young larvae (no adults) induced by food limitation and crowding worms release a pheromone under these conditions; induces dauer stress resistant delays reproduction

Can survive long time on dauer state Physiologic effects similar to CR When food becomes available, the worms leave the dauer stage, become sexually mature, and have offspring. Clear survival mechanism

Daf-2 (Dauer formation 2) gene The Daf-2 gene regulates entry into dauer stage. mediates endocrine signaling, metabolism increased signaling arrests development in worms inducing a dauer state

Daf-2 mutations extend lifespan Kenyon and colleagues at UCSF found three mutations in the Daf-2 gene (sa189, sa193, and e1370) that greatly extended the worm’s lifespans Mutations did not put worms into dauer stage.

Mean lifespan for wild type worms was 18 days. Mean lifespan for daf-2 (sa189) mutant was 42 days. When all the wild-type animals were dead or immobile, 90% of the daf-2 (e1370) mutants still moved actively. Daf-2 mutants were not stalled in dauer stage; they became full-size adults that behaved normally, except for slightly smaller than normal brood sizes.

How do daf-2 mutations extend lifespan? The gene daf-16 acts downstream of daf-2 to promote dauer formation. Mutations in daf-16 completely block the effects of daf-2 mutations, meaning that a functioning daf-16 is required for extended lifespan. Identification of C elegans genes that act downstream of daf-16 could lead to a general understanding of how lifespan can be extended.

Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans. Murphy CT, McCarroll SA, et al Kenyon lab, UCSF

Background Ageing is regulated by a conserved insulin/IGF-1 signaling pathway (see results in mice in next lecture). C. elegans normally lives a few weeks, but mutations that decrease insulin/IGF-1 signaling, such as daf-2 insulin/IGF-1 mutants, remain youthful and live twice as long as normal. The daf-2 pathway regulates reproduction, lipid metabolism, and dauer formation independently of each other. For example, during development it regulates dauer formation, but in adulthood it acts exclusively to influence aging.

Daf-2 mutations require functioning daf-16 to extend lifespan. Daf-16 is a FOXO-family transcription factor (regulates expression of other genes) It should be possible to learn how insuling/IGF-1 signaling influences aging by identifying and characterizing the genes regulated by daf-16. Animals with reduced daf-2 activity are resistant to oxidative stress, suggesting an increased ability to prevent or repair oxidative damage (damage from free radicals).

Methods Kenyon and colleagues studied gene regulated by daf-16 using two methods: Microarrays measure gene expression. RNA interference (RNAi) prevents a gene from producing its corresponding protein, similar in effect to a knock-out.

Results Found two classes of genes 1. Genes induced in daf-2 mutants but repressed in daf-16 RNAi animals. These are candidates for genes that extend lifespan. 2. Genes with the opposite profile, which are candidates for shortening lifespan.

stress response genes Class 1 (possible lifespan extension) included genes for increased stress response (genes that prevent or repair damage from free radicals). Kenyon inactivated these genes using RNAi, and found that lifespan was shortened, up to 20%

Class 1 also included genes that protect against bacteria, which the worms eat, but which eventually overwhelm and eat the worm. Kenyon inactivated these genes using RNAi, and found that lifespan was shortened. These results confirmed that the genes upregulated by daf-16 promote longer lifespan.

Comments from Kenyon Longevity must have evolved not just once, but many times. Evolutionary theory postulates that lifespan is determined by the additive effects of many genes, consistent with our findings. The beauty of the insulin/IGF-1 system is that it provides a way to regulate all of these genes coordinately. As a consequence, changes in regulatory genes encoding insulin/IGF-1 pathway members or daf-16 homologs could, in principle, allow changes in longevity to occur rapidly during evolution.

Daf-2, Insulin Receptor-like Gene in Worms Kimura, Tissenbaum, Liu, Ruvkun

Do humans have a gene like daf-2 that may control lifespan? The daf-2 protein is most similar to two closely related human receptors, the insulin receptor (IR) and the insulin-life growth factor receptor (IGF-1R).

Daf-2 is the only member of the insulin receptor family in the worm’s genome. Daf-2 is equally distant from human receptors (35% identical to human) and is probably a homolog of their ancestor. So it may subserve any or all of their functions.

Daf-2 and the human insulin receptors both regulate metabolism. A human diabetic insulin-resistant patient has the same amino acid substitution found in a mutant daf-2. (Pro1178  Leu) This 14 year old was morbidly obese suggesting that effects of decreased insulin signaling were similar to daf-2 mutants.