The serious search for an antiaging pill'

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The serious search for an anti-aging pill.

• Mark Lane, Donald Ingram George Roth

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• 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.

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• 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?

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• 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.

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• 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.

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• 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

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• 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

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• 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.

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• 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.

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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.

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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.

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Nutritional Control of Aging

• Zimmerman, Malloy, Krajcik-May

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Hypothesis

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

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Materials

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

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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.

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Results of MR diet

• significantly stunted growth
• extended life by 42 mean and 46 maximal
  longevity
• MR ate less food than CR.

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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

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A C. elegans mutant that lives twice as long as
wild type.

• Kenyon C, Chang J, Gensch E, Rudner A, Tabtiang R.

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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

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• 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

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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

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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 worms
  lifespans
• Mutations did not put worms into dauer stage.

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• 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.

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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.

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Genes that act downstream of DAF-16 to influence
the lifespan of Caenorhabditis elegans.

• Murphy CT, McCarroll SA, et al
• Kenyon lab, UCSF

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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.

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• 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).

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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.

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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.

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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

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• 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.

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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.

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Daf-2, Insulin Receptor-like Gene in Worms

• Kimura, Tissenbaum, Liu, Ruvkun

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• 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).

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• Daf-2 is the only member of the insulin receptor
  family in the worms 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.

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• 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.