L7, Dietary restriction

Question 1

How does dietary restriction affect organisms lifespan and fecundity?

Answer 1

• Reduced food intake without starvation or malnutrition results in extended lifespan and late life health
• Reproduction, however, gets worse as relative caloric intake reduces (reduced fecundity)

Question 2

How are the effects of DR measured?

Answer 2

• Survival analysis to measure the lifespan of a population of animals -> often median lifespan used
• Analysis of health and function throughout life -> fecundity/reproduction, immune function, locomotor function, incidence of age related disease (obesity, cancer, CHD etc)

Question 3

Results of DR studies in flies:

Answer 3

• Lower fecundity in DR flies (measured by rate of egg laying)
• Increased lifespans in both females and males (bigger effect in females than males) -> sexual dimorphism
• Dietary restriction with supplementation of essential amino acids restored normal lifespan (both median lifespan and survival analysis); also restored fecundity

Question 4

DR in rodents: 3 methods

Answer 4

1. Control animals given unrestricted diet (‘ad libitum’) and DR animals are given a percentage of the AL diet
2. Control animals are fed an average daily amount of food and the DR animals are fed a percentage of this amount
3. Intermittent fasting at every other day (OED) feeding

• Important to remember that specific AAs are modulating the DR lifespan effect
• Aiming the control to be a true representation of healthy mice; ad libitum is ill-advised as obese mice have their own mortality risks associated and DR would relieve this

Question 5

DR in Rhesus monkeys:

Answer 5

• Improved survival
• Reduced incidence of cancer, CD, diabetes, obesity prevention

Question 6

DR in human volunteers:

Benefits

Answer 6

• Beneficial effects against obesity, diabetes, insulin resistance, inflammation and oxidative stress
• -> Reduced risk factors for CD and cancer

Question 7

+ Hallmarks of DR:

Impact on major causes of death?

Answer 7

• CD disease (lower, with better post-ischemic recovery)
• Excess adiposity and diabetes (lowered, with better insulin sensitivity and glucose tolerance)
• Kidney disease (lower, with better glomerular filtration)
• Autoimmune disease and inflammation (lowered, with better SC self-renewal)
• ND disease (lower amyloid deposition, atrophy and inflammation in brain)
• Cancer (lower with later onset, better genetic stability)

Question 8

Side effects of DR in humans:

Answer 8

• Can lead to infertility, sarcopenia, osteoporosis, immune deficiencies
• Long term effects on resistance to infection, wound healing and cognitive function not known

Question 9

Evidence for the IIS/TOR signalling network mediating the lifespan effects of DR:

Answer 9

• Overall: response of lifespan to DR is altered or removed in animals mutant for IIS/TOR signalling
• The lifespan of TOR mutants in flies and worms cannot be extended further by DR
• Deletion of DILPs or ablation of IPCs (i.e. reduced IIS) in flies protects them from the effects of high yeast intake
• Overexpression of FOXO in flies produced an altered response to DR
• The median lifespan of GHR KO mice is not extended further by DR

Question 10

Describe the AMPK protein and its involvement in IIS/TOR signalling pathway:

Answer 10

• AMPK is considered a key checkpoint to ensure energy balance in both cells and organisms (‘nexus’)
• Negatively regulated by pAKT, S6K
• Active AMPK activates ULK1 -> autophagy
• Active AMPK activates FOXO
• Heterotrimer controlled by allosteric regulation by AMP, ADP and ATP, and phosphorylation
• Acts as a protective response to energy stress, and is a key player in diabetes and related metabolic diseases and cancer, as well as aging

Question 11

Evidence in flies and worms for role of AMPK in lifespan extension:

Answer 11

• Activating AMPK is sufficient to extend lifespan in model organisms
• e.g. Increasing expression of aak-2 in C.elegans increases lifespan by 13% and mimics DR in well-fed animals
• Overexpression of the single Drosophila AMPK-alpha subunit in either muscle or the fat body extends lifespan in the fruit fly

Question 12

Early findings on role of sirtuins in aging:

Answer 12

• Respond to NAD/NADH ratio
• Activate FOXO (removing inhibitory modification)
• First elucidated in budding yeast -> overexpression of SIR2 increased replicative lifespan
• Later shown that increased levels of sirtuins in worms and flies also extends lifespan -> evolutionarily conserved role?

Question 13

Early findings on resveratrol:

Answer 13

• Purported to be a pharmacological activator of sirtuins; promoted as a therapeutic agent in humans to mimic DR and slow aging
• -> recent studies raise questions about the evolutionary conservation of the role of sirtuins in lifespan

Question 14

Outline sirtuins in yeasts vs mammals:

Answer 14

• First described as NAD-dependent type II histone deacetylases (Sir2)
  -> silences specific loci in DNA via deacetylation of H3 and H4
• Mammalian sirtuins (1-7) deacetylase histones and other proteins in other cellular compartments
• -> also function as ADP-ribosyltransferases

Question 15

Evidence for role of sirtuins in lifespan:

Answer 15

• Increased expression of sir-2 gene in worms extended lifespan
• Increased dsir2 in flies extended lifespan, decreased expression blocked effect of DR
• Sirt1-/- mice do not show lifespan extension under DR conditions

Question 16

Evidence against role of SIRT1 in lifespan:

Answer 16

• Overexpression of Sirt1 in mice did not extend lifespan but these mice showed reduced cancer risk and were protected from metabolic dysfunction
• Mice show lifespan extension with resveratrol but only under high fat diets (i.e. resveratrol was rescuing the negative effects of this diet)
• Later studies in worms and flies failed to replicate the lifespan extension effects of Sir2 overexpression or resveratrol

Question 17

Regulators of sirtuin activity (x3):

Answer 17

• Diet and metabolism: High energy represses, low energy activates; via TFs and via NAD+ substrate levels
• Oxidative stress: high ROS levels activate JNK which in turn phosphorylates and activates SIRT1
• Genotoxic stress (e.g. UV) inactivates sirtuins

Question 18

+ Key targets for geroprotective signalling pathways

Answer 18

• AKT
• FOXO
• mTOR
• Nicotinamide adenine dinucleotide (NAD+)
• AMP-activated protein kinase (AMPK)
• Fibroblast growth factor 21 (FGF21)

Question 19

How is DR achieved in flies?

Answer 19

• Achieved by diluting the concentration of food medium -> either total food or reducing levels of specific amino acids
• Food medium is sugar and yeast in an agar jelly
• Specific amino acids mediate lifespan and fecundity changes under DR

Question 20

2 examples of fly DR protocol issues:

Answer 20

• Salt toxicity in Brewer’s yeast food medium -> DR rescuing deleterious effect
• Sugar can betray fecundity levels, confusing effects

Question 21

Changes enacted by DR (initiating hallmarks of DR):

Answer 21

• Transcriptome and epigenomic changes
• Proteome, metabolome and gut microbiome modifications
• Reduced accumulation of molecular and cellular damage
• -> Increased healthspan and extended lifespan

Question 22

Overview of DR regulating IIS/TOR:

Answer 22

• DR affects IIS/TOR network, inhibiting both
• This is a major way in which DR mediates lifespan extensions
• FOXO nuclear import -> increased level of proteins that repair and protect cell (proteostasis, autophagy and mitophagy, DNA repair, stem cell self-renewal etc)

Question 23

Possible harmful effect of AMPK signalling:

Answer 23

• Often dysregulated in cancer/ND disease
• -> possible oncogenic role in some circumstances due to energy balance promoted
• Lack of complete understanding m