Dauer diapause in C. elegans is a nonfeeding, stress resistant larval state whose purpose is to provide endurance for dispersal under adverse conditions. When overpopulation leads to scarcity of food (bacteria), worms stop feeding and a pheromone forces them to enter the “Dauer” phase. In this state, worms can live for two months or longer. On the other hand, when there is enough food, the worms die after only two weeks. In this situation, in the absence of a pheromone (permissive growth conditions), an insulin-like ligand activates the DAF-2 receptor (29).

daf-2 encodes an insulin-receptor-like protein (the worm ortholog of the insulin/IGF-1 receptor gene in mammals) which when activated promotes the recruitment of the product of the age-1 to the plasma membrane (3).

age-1 encodes the worm ortholog of the phosphoinositide 3-kinase (PI3K) p110 catalytic subunit (AGE-1/PI3K). PI3K recognizes and phosphorylates phosphatidylinositol-4, 5-bisphosphate (PIP2) generating the membrane-localized signaling molecule, phosphatidylinositol-3, 4, 5-triphosphate (PIP3). DAF-18 (the worm ortholog of the mammalian PTEN) antagonizes AGE-1 by dephosphorylating the inositol ring in the third position (converting PIP3 back into PIP2) (5).

PIP3 activates PDK-1 kinase. PIP3 together with PDK-1 activity are necessary for the activation of the AKT-1 and AKT-2 kinases (11).

AKT-1 bears two phosphorylation sites (corresponding to Thr-308 and Ser-473 in the human ortholog Akt/PKB, which are necessary for its activation).
AKT-2 only has the phosphorylation site equivalent to Thr-308 in human PKB.
In mammals, Akt/PKB is phosphorylated at Thr-308 by PDK1 and at Ser-473 by the as yet unpurified PDK2. Therefore AKT-1 may couple to a PDK1 and PDK2-like kinase whereas AKT-2 may couple to only PDK1 (10).

Simultaneous inhibition of both AKT-1 and AKT-2 activities using RNA interference (RNAi) causes nearly 100 % arrest at the dauer stage, whereas inactivation of either gene alone does not, showing that their function is redundant (10).

Activated AKT-1 and AKT-2 inhibit the activity of forkhead transcription factor DAF-16 most likely by phosphorylation (10), preventing its accumulation in the nucleus (12).

DAF-16 contains four consensus sites for phosphorylation by Akt/PKB, and three of these sites are conserved in the human DAF-16 orthologs AFX, FKHR, and FKHRL1 (9,10).

Mutations in daf-16 completely suppress the dauer arrest and metabolic shift of animals bearing daf-2, age-1, or pdk-1 mutations or RNAi inhibited AKT-1 and AKT-2 activity (9). daf-16 mutations also suppress the increase in longevity caused by decreased DAF-2, AGE-1, or PDK-1 signaling (9). Thus, DAF-16 is active in the absence of these upstream inputs and acts to increase life span.

DAF-16 action regulates a wide range of physiological responses by altering the expression of genes involved in metabolism, energy generation and cellular stress responses (13).

DAF-16 and its mammalian orthologs bind to an identical consensus DNA sequence (TTGTTTAC) in vitro (16).

Several gene products have been identified downstream of DAF-16:

1) Superoxide dismutase 3 (SOD3), which is a mitochondrial manganese superoxide dismutase, is an important enzyme that protects against the reactive oxygen species (ROS) superoxide anion (18, 19).
NOTE: SOD3 in C. elegans is equivalent to the SOD2 in other organisms.

2) Metallothionein (MT1), which is a small cysteine-rich metal binding protein, is implicated in protection against heavy metal toxicity and ROS associated damage. C. elegans has two forms of Metallothioneins: Metallothionein-1 (MT1) is constitutively expressed in three cells of the posterior bulb of the pharynx but is also induced by Cd and heat in intestinal cells. MT2 mRNA is not expressed under basal conditions and occurs only in intestinal cells, where it is induced by Cd.
daf-2 mutation results in increased Cd and Cu ion resistance comparing with the WT worms, which correlated with an increase in MT1 mRNA levels in the daf-2 mutant (20).

3) The heat shock proteins HSP70, HSP90, HSP-16 (3), which are involved in the reparation of missfolded or damaged proteins and are essentials for the recovering of the cells after a heat treatment. Interestingly, the overexpression of HSP70F, a member of the HSP70 family, produces an increase in longevity (21), indicating that protein missfolding and aggregation may be an important factor in aging as it is for heat stress sensitivity.

4) OLD-1 is a transmembrane tyrosine kinase protein that is upregulated in the long-lived daf-2 and age-1mutants in a daf-16 dependent manner (3). Its function is necessary for the increase of longevity of daf-2 and age-1 mutants (22); furthermore old-1 overexpression produces a substantial increase of longevity, congruent with a positive regulatory role for life span. Interestingly, this gene also positively regulates stress resistance, since OLD-1 overexpression produces an increase of stress resistance and its mutation makes animals more sensitive to UV and heat stress. The expression of old-1 is not only upregulated in a long-lived mutant background but also under stressful conditions (22). The fact that old-1 encodes for a tyrosine kinase indicates that it should have a regulatory function more than a direct role in the reparation of damage caused by stress (3).

5) SLC-1 is a putative secretory protein with an SCP domain and is homologous to the mammalian cysteine-rich secretory protein (CRISP) family. slc-1 is required for the extension of the life span of daf-2 and age-1 mutants, and downregulation of slc-1 reduces both life span and stress resistance of this animal (17).

6) ZK384.3 is a predicted non-proteosomal aspartyl protease which when targeted by RNAi reduced the mean life span of daf-2 mutant from 44.5 days to 29.5 days (13).

7) PNK-1 which is one of the two pantothenate kinases in C. elegans and is up-regulated in the daf-2 mutant (14). Pantothenate kinases are the rate limiting enzymes in coenzyme A synthesis (14).

NOTE: Genetic studies demonstrate that two parallel pathways control dauer formation. One is regulated by the transforming growth factor ß (TGF-ß) signaling pathway and the other by the IGF/insulin-like signaling pathway. Single mutations in either pathway can induce dauer formation in conditions that are good for growth to adult, but only mutations affecting the insulin pathway have increased longevity in the adult stage (3).

Figure 1. The insulin-like signaling pathway that controls development/reproductive growth and dauer state/increased longevity in C. elegans. (Adapted from (29))
A) During favorable growth conditions, unc-64 and unc-31 function to promote an as yet unidentified insulin-like ligand (INS) that binds to the DAF-2 receptor. This signaling cascade results in the repression of the DAF-16 transcription factor activity and the entrance into a state of growth and short life span.
B) Under starvation conditions, there is no release of the insulin-like ligand resulting in activation of DAF-16 which accumulates in the nucleus and transcribe genes that provide stress-resistance and increased life span.

INSULIN-LIKE GROWTH FACTOR 1 SIGNALING CASCADE IN MAMMALS

Mammalian insulin/insulin-like growth factor 1 (IGF-1) signaling is involved in cellular growth, proliferation, metabolism and survival (1). The insulin receptor (IR) and the insulin-like growth factor receptor (IGFR) are receptor tyrosine kinases that become activated upon insulin and IGF-1 binding, respectively (1).

Activation of the insulin/IGF-1 receptor by insulin/IGF-1 causes the receptor to phosphorylate itself at several tyrosine residues.

The phosphorylated tyrosines recruit the insulin receptor substrate (IRS1 – IRS4) adaptor proteins. These adaptor proteins contain a phosphotyrosine binding (PTB) domain which binds to a tyrosine-phosphorylated juxtamembrane consensus motif (NPxY) on the IR and IGF-1R.

The IRS adaptor proteins also contain multiple tyrosine and serine/threonine (S/T) phosphorylation sites in their large C-terminal segment (1) The IR/IGFR phosphorylate several of these tyrosines on the adaptor proteins, specially those present in a YxxM consensus motif. These phosphorylated tyrosines are recognized by the SH2 domains of the p85 subunit of the enzyme phosphoinositide 3 –kinase (PI3K) that is recruited to the plasma membrane.

The p85 PI3K subunit (adaptor) binds to the class 1A p110 PI3K catalytic subunit, which by virtue of being recruited to the plasma membrane catalyzes the conversion of phosphatidylinositol- (4,5)-bisphosphate (PIP2) into phosphatidylinositol- (3,4,5)-triphosphate (PIP3) by phosphorylating the D-3 position in the inositol ring.

The action of the PI3K is opposed by the 3’ lipid phosphatase tensin homolog (PTEN), which catalyzes the conversion of PIP3, into PIP2 (4).

PIP3 interacts with and activates the phosphoinositide-dependent kinase 1 (PDK1) (2).
PDK1 partially activates the serine/threonine kinase “protein kinase B” (PKB) by phosphorylating it at Thr308.

PKB contains an N-terminal pleckstrin homology (PH) domain. Maximal activation of PKB also requires PIP3 binding to its PH domain and the phosphorylation of Ser473 on PKB by an unknown kinase. The insulin-induced phosphorylation of Ser473, like the phosphorylation of Thr308 is prevented by inhibitors of PI3K, suggesting that Ser473 may be phosphorylated by a distinct 3-phosphoinositide-dependent protein kinase (2), which is referred to as PDK2.
PKB, also known as AKT1, directly phosphorylates the forkhead box, class O transcription factors (collectively known as FOXO), AFX (acute-lymphocytic-leukaemia-1 fused gene from chromosome X), FKHR (Forkhead in rhabdomyosarcoma) and FKHRL1 (FKHR-like 1) inhibiting their transcriptional activity (6, 7, 8).
These transcription factors are nuclearly localized only when insulin-like signaling (AKT activity) is low (9). Otherwise these transcription factors get phosphorylated and they are retained in the cytosol, where they are inactive (15).

Studies have shown that this evolutionarily conserved signaling pathway is similar to that of C. elegans regarding the increase in oxidative stress resistance since overexpression of FKHRL1 showed an increase in hydrogen peroxide scavenging capacity (23). Likewise, in the absence of PKB activity, FKHRL1 increases the expression of MnSOD (24).
It has also been shown that FKHRL1 functions at the G2 to M checkpoint in the cell cycle and triggers the repair of damaged DNA through the Gadd45 (a protein with cyclin-dependent protein kinase inhibitory activity involved in DNA repair and growth arrest) (25).

Figure 2. Proposed mechanism by which IGF-1 induces the inhibition of the FOXO transcription factors. See text for details.

IGF-1 vs. INSULIN SIGNALING IN MAMMALS

Insulin-like growth factor 1 (IGF-1), also known as somatomedin C, is a protein of 70 aminoacids whose synthesis is regulated by growth hormone (GH), insulin and nutritional intake (36). Although GH is the key regulator of IGF-1 gene expression and peripheral IGF-1 levels, some IGF-1 is synthesized independently of GH. IGF-1 is widely synthesized in several organs including liver, bones, muscle, brain, intestines, and gonads, but the majority of circulating IGF-1 is derived from the liver (36).

NOTE: It may be the case that the circulating IFG-1 (which acts at the endocrine level and is synthesized by the liver) differs from the IGF-1 that is synthesized by other organs and acts locally (at the paracrine and autocrine level). Apparently this is the case at least for the IGF-1 isoform that acts at the level of muscle.

IGF-1 circulates in plasma as a complex with IGF binding proteins (IGFBP) and acid labile subunit (ALS), as well as in free form. Because only free IGF-1 is believed to be available for interacting with its receptors and exerting its physiological effects, IGFBPs, six of which have been identified, and proteases that cleave IGFBPs into smaller fragments incapable of binding IGF-1, are important modulators of IGF-1 actions (34).

IGF-1 acts primarily by binding to its cognate IGF-1 receptors (IGF-1R) but it can signal also via insulin receptor. In the same way, insulin and IGF-II can also bind to IGF-1R (34). Besides this overlap in ligand activation, the IGF-1R and the insulin receptor (IR) are similar in structure and activate many of the same post-receptor signaling pathways. However; there is evidence that these two ligands (IGF-1 and insulin) mediate different biological functions. It has been shown that in mouse fibroblasts several genes that are upregulated by IGF-1 are not upregulated by insulin and vice versa (35) so there is specificity in the signaling of each ligand.
The difference between the post-receptor signaling is illustrated by the phenotypes of mutant mice. Mice lacking IR are born with modest growth retardation (~10%). Their embryonic development is otherwise unimpaired. After birth, they rapidly develop diabetic ketoacidosis and die within a few days. IGF1-R-deficient mice are severely growth retarded (~ 45% of normal) and die within minutes of birth, probably as a result of respiratory failure caused by impaired development of the diaphragm and intercostals muscles (37). From these studies and others it is established that the principal actions of IGF-1 in mammals concern control of growth and organ size via mitotic and anti-apoptotic effects, but its physiological role includes participating in glucose homeostasis and potentiation of action of other hormones via modifying levels of their respective receptors, etc. (34).

IGF-1 SIGNALING AND AGING IN MAMMALS

Studies in Caenorhabditis elegans demonstrate that disruption of the daf-2 signaling pathways extends lifespan. Similarities among the daf-2 pathway, insulin-like signaling in flies and yeast, and the mammalian insulin-like growth factor 1 (IGF-1) pathway raise the possibility that modifications to IGF-1 signaling could also extend lifespan in mammals.

In fact, the Ames and Snell dwarf mice, which have mutations in the Prop-1 and Pit-1 loci required for pituitary development, live approximately 50% longer than wild-type mice (26). These dwarf mice are deficient in three pituitary hormones: growth hormone (GH), prolactin and thyroid-stimulating hormone. The GH deficit causes a decreased circulating IGF-1 and insulin levels, which is believed to be the cause of increased longevity in these strains (26, 27). Consistent with this theory, disrupting the gene encoding the GH-receptor (in the Laron dwarf mice) causes high plasma GH levels, low plasma IGF-1 levels (due to impaired GH signaling) and a life span increase of 47% (26). In addition, the lit mutant mouse strain, which has a mutation disrupting the hypothalamic GH releasing hormone (GHRH), also lives longer. Homozygous lit/lit mice live up to 25% longer than wild-type mice (26).

Homozygous knockout mice for the Igf1 receptor (Igf1r -/-) are not viable (32); however; heterozygous knockout mice for this receptor (Igf1r +/-) are viable and live 26 % longer than the wild type (32). Moreover, these mice do not develop dwarfism, and have a normal energy metabolism, nutrient uptake, physical activity and fertility (32). Consistent with IGF1 signaling disruption in other organisms, these mice also display greater resistance to oxidative stress (32). It has also been shown than mice with a fat-specific insulin receptor knockout (FIRKO) have an increase in mean life-span of 18% with parallel increases in median and maximum life-spans (33).

Mice overexpressing GH (i.e. supraphysiological plasma levels) exhibit a 50% reduction in life span, significantly reduced levels of antioxidative defense molecules and increased oxidative damage to cellular constituents (27). Significant indicators of premature aging observed in these mice include glomerulosclerosis, reduced replicative potential, increased expression of GFAP (glial fibrillary acidic protein), reduced neurotransmitter turnover, early loss of reproductive competence and exacerbated age-related declines in cognitive function (learning and memory) (27).

This information suggests that the extension of longevity mediated by absence of the IGF1 signaling pathway is conserved through evolution in different species. The mediators of this effect seem to be the forkhead transcription factors which are negatively regulated by this pathway in all species. The increase in stress-resistance mediated by these transcription factors may be the reason why longevity is extended.

Figure 3. Adapted from reference (28). Wild-type (left specimen) and long-lived dwarf (right specimen) yeast, flies, and mice with mutations that decrease glucose or insulin/IGF-I-like signaling. Yeast sch9 null mutants form smaller colonies (left). sch9 mutants are also smaller in size, grow at a slower rate, and survive three times longer than wild-type yeast. chico homozygous mutant female flies are dwarfs and exhibit an increase in life-span of up to 50% (center). Chico functions in the fly insulin/IGF-I-like signaling pathway. The GHR/BP mice are dwarfs deficient in IGF-I and exhibit a 50% increase in life-span (right). Other yeast and worm mutants exhibit life-span extension of more than 100% but do not have detectable growth defects.

CALORIC RESTRICTION AND IGF-1 SIGNALING

Caloric restriction (CR), which is a restriction in the number of calories consumed daily, extends longevity in organisms from yeast to mice (29, 30). CR also postpones or prevents a number of diseases and age-dependent deterioration without causing irreversible developmental or reproductive defects (28).

In yeast, worms and flies, the partially conserved glucose or insulin/IGF-1 like pathways down-regulate antioxidant enzymes and heat shock proteins, reduce the accumulation of glycogen or fat, and increase growth and mortality (28). Mutations that reduce the activity of these pathways appear to extend longevity by simulating CR or more severe forms of starvation (28). In mice and humans, this pathway seems to be conserved, but further studies have to be performed to understand the downstream mediators of the life-span extension effect in these organisms. A common downstream candidate could be the MnSOD, which is downstream of DAF-16 and its orthologs in yeast (31), worms (13, 18, 19), and human cells (24).

CALORIC RESTRICTION MIMETICS AS POSSIBLE ANTI-AGING INTERVENTIONS

Even though CR retards diseases and aging in all species tested so far, it is unlikely that most humans would be willing to maintain a 30% reduced diet for the bulk of their adult life span, even if it meant more healthy years. For this reason, scientists have begun to explore CR mimetics, agents that might elicit the same beneficial effects as CR, without the necessity of dieting.
The associations between changes of glucose metabolism accompanying CR and its longevity effects suggest the following possibilities for metabolic mechanisms:

(a) lowering of glucose levels;
(b) lowering of insulin levels;
(c) enhancement of insulin sensitivity;
(d) some combination of these.

These various aspects of glucose and insulin regulation can be affected by a number of experimental interventions, including drugs that lower plasma glucose or insulin levels, or raise insulin sensitivity. Candidate compounds include the following:

• Biguanides (example: metformin). Although these agents have been available for nearly 30 years, their exact mechanism(s) of action is still uncertain. Their biological effects include lowering blood lipids, reducing gluconeogenesis (the opposite of CR), decreasing glucose adsorption through the intestine and very importantly facilitating the entry of glucose into cells, which results in increased insulin sensitivity (like CR) (40).

• Sulfonylureas (example: glyburide). These drugs lead to mild to moderate hypoglycemia in experimental animals apparently by enhancing insulin secretion and pancreatic beta cell sensitivity to stimuli. Insulin levels would likely be similar to those of control (but high for the circulating glucose level) or possibly mildly elevated compared with controls. Insulin sensitivity may or may not be affected in such a model (39).

• Thiazolidinediones (example: troglitazone). The precise mechanism of action of these drugs is also unknown, they appear to regulate certain genes involved in both lipid and carbohydrate metabolism and, like the biguanides, increases glucose entry into cells (40).

• Alpha-glucosidase inhibitors (example: acarbose). These drugs work by inhibiting glucose absorption from the gastrointestinal (GI) tract by interfering with digestion of complex carbohydrates in the diet. Their primary effect is to lower post meal glucose levels. There is no direct effect on insulin action or insulin secretion (39).

• Phlorizin. This drug lowers the renal threshold for glucose excretion, thereby increasing glucose loss. Glucose levels in experimental animals would likely be lowered, without direct effects on insulin sensitivity (39).

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