Genetic Pathways of Postmitotic Cell and Organismal Survival

Besides the role of a potential ectopic cell cycle checkpoint pathway helping to modulate the life span of a postmitotic organism such as C. elegans, are there other

WT + Diet Restriction

WT + Reduced Insulin Signaling (IIS)

WT + Diet Restriction + Reduced IIS

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WT + Reduced Insulin Signaling (IIS)

WT + Reduced ETC + Reduced IIS

WT + Diet Restriction

WT + Reduced Insulin Signaling (IIS)

WT + Diet Restriction + Reduced IIS

WT + Reduced ETC

WT + Reduced Insulin Signaling (IIS)

WT + Reduced ETC + Reduced IIS

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Fig. 1.2 Reduced insulin/IGF-1 signaling (IIS) synergizes with perturbations in other longevity pathways. Representative life span analysis of worms with combinatorial inactivation of IIS, diet restriction, or reduced mitochondrial electron transport chain (ETC) activity is depicted. A) Combination of diet restriction (depicted as red line) with reduced IIS (green line) results in animals that live longer than either single perturbation (blue line). B) Combination of reduced ETC (depicted as yellow line) with reduced IIS (green line) results in animals that live longer than either single perturbation (cayenne line). For more details of separation of these three pathways, please see text (See Color Plate)

Time

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Fig. 1.2 Reduced insulin/IGF-1 signaling (IIS) synergizes with perturbations in other longevity pathways. Representative life span analysis of worms with combinatorial inactivation of IIS, diet restriction, or reduced mitochondrial electron transport chain (ETC) activity is depicted. A) Combination of diet restriction (depicted as red line) with reduced IIS (green line) results in animals that live longer than either single perturbation (blue line). B) Combination of reduced ETC (depicted as yellow line) with reduced IIS (green line) results in animals that live longer than either single perturbation (cayenne line). For more details of separation of these three pathways, please see text (See Color Plate)

genetic pathways that determine longevity and survival of postmitotic cells? And, if so, how do they impact genome integrity?

Surprisingly, at least three distinct pathways have been found to promote survival of worms, flies, and mice. These pathways include the insulin/IGF-1 signaling pathway (IIS), dietary restriction pathway (DR), and mitochondrial electron transport chain (ETC) pathway (Fig. 1.2). Below, we have outlined the important discoveries of each pathway and explain in greater detail their independence from each other.

5.3.1 Insulin/IGF-1 Signaling

Well over 15 years ago, single-gene mutations in worms were identified that extended longevity. One such mutation, age-1, identified by Michael Klass, turned out to be the vertebrate ortholog of the phosphoinositide 3-kinase essential for transduction of signals from the insulin receptor to downstream kinases, such AKT (Klass 1983, Morris et al. 1996). Since this discovery, mutations in genes for all known members of the insulin/IGF-1 signaling (IIS) pathway have been found to extend longevity of adult worms (reviewed in Wolff and Dillin 2006). Furthermore, all increases in longevity induced by mutations in the IIS depend upon the forkhead-related transcription factor DAF-16, the worm ortholog of the vertebrate Foxo3a. Mutating chico, the fly ortholog of IRS-1, reduces signaling along the IIS pathway and increases longevity in flies. Similarly, reducing expression levels of the IGF-1 receptor (IGF-1R), or knocking out the insulin receptor in adipose tissue, extends longevity of mice. Taken together, reduction of IIS in many distinct organisms extends longevity, suggesting that the IIS pathway plays a highly conserved role in cell survival. Interestingly, knockout of the yeast AKT ortholog, Sch9, extends the replicative life span of yeast cultures (Fabrizio et al. 2001), suggesting that the IIS pathway could potentially upregulate survival cues in both postmitotic and mitotic cells.

How does reduced IIS extend longevity? One possibility is that the IIS pathway feeds back into the DNA damage surveillance pathway and possibly even the regulation of telomere length. DNA microarray analysis of genes regulated by DAF-16 suggests that the IIS pathway (Murphy et al. 2003) intersects with the DNA damage pathway and induces the expression of genes required for resistance to DNA-damaging agents. Consistent with this analysis, IIS mutants are highly resistant to DNA-damaging agents (Larsen 1993). However, a connection of IIS with telomere maintenance is lacking. Analysis of telomere length regulation in animals with reduced IIS, or deletion of DAF-16, found no effect, excluding telomere length regulation as a possible means through which IIS mutant animals extend their life span (Raices et al. 2005). In addition to increased expression of DNA repair and surveillance genes, the IIS pathway also regulates expression of genes required for proper protein folding, innate immunity, and oxidative stress (Murphy et al. 2003). Therefore, in addition to DNA damage surveillance a fine-tuned execution of many different stress-responsive modules is required for a successful increase in survival.

5.3.2 Dietary Restriction

Over 72 years ago it was discovered that reducing food intake results in increased longevity, but the underlying molecular mechanisms have yet to be uncovered. A priori, it would appear that the mechanism by which reduced insulin/IGF-1 signaling mediates a response to increased cell survival should be consistent with the response to reduced food intake. However, many pieces of data suggest otherwise. First, the long life span induced by diet restriction in worms does not depend upon DAF-16 (Houthoofd et al. 2003, Lakowski and Hekimi 1996). Second, long-lived insulin/ IGF-1 mutant worms have an even greater life span extension when maintained on a diet-restricted regimen (Lakowski and Hekimi 1996). Third, dietary restriction can be instituted at almost any time in the animal's life cycle, whereas insulin/IGF-1 signaling is required during the early adulthood stages in order to regulate longevity cues (Dillin et al. 2002). Finally, many of the physiological outcomes of animals with reduced IIS compared to animals undergoing DR are similar. Such overt similarities include reduced body size, lower plasma IGF-1 and insulin levels, and increased insulin sensitivity. Furthermore, transcriptional profiling of long-lived dwarf mice with reduced IGF-1 signaling found additional increased expression of multiple liver-specific genes when combined with dietary restriction (Tsuchiya et al. 2004). However, compelling genetic analyses indicate that many key differences among IIS and DR mice exist as well. For example, IGF-1R long-lived heterozygous mice do not show protracted or reduced reproduction as DR animals do (Holzenberger et al. 2003).

To date, the only protein directly implicated in the longevity response to DR is the histone deacetylase, SIR-2. This hypothesis stems from the fact that ectopic expression of SIR-2 extends longevity of yeast, worms, and flies. Furthermore, yeast defective in sir-2 cannot respond to increased longevity due to growth on low glucose conditions (glucose restriction) (Lamming et al. 2005). However, deletion of sir-2 does not disrupt a worm's ability to live long under conditions of diet restriction (Wang and Tissenbaum 2006). Furthermore, and most compelling, overexpression of sir-2 in worms results in increased longevity that is dependent upon daf-16 (Tissenbaum and Guarente 2001). Taken together, it is not clear where exactly sir-2 fits into longevity pathways, however, it is interesting to speculate that the role of sir-2 in telomeric DNA silencing in yeast may be conserved with telomere maintenance in other organisms and thus could account for the longevity increases associated with overexpression of sir-2 (Moretti et al. 1994). The role of sir-2 and its paralogs in mice will reveal much about the role of protein deacetyla-tion in longevity determination.

5.3.3 Mitochondrial Electron Transport Chain

Mitochondria have been implicated in the aging process for several decades. Measuring the metabolic rates of several species during the 1920s, Pearl discovered the correlation between metabolic rate and life span: animals with lower metabolic rates lived longer than animals with higher metabolic rates (Pearl 1928). Exceptions have been discovered since Pearl's initial observations, such as the high metabolic rates of some long-lived birds (Holmes et al. 2001); however Pearl's initial observation led to the formulation of the "rate of living theory of aging." The theory suggests that reduced metabolic rates in an animal should result in an increased life span.

Several years later, the "rate of living theory of aging" became synonymous with the "oxygen radical theory of aging" proposed by Denham Harman (Harman 1956; see also Passos et al., this volume). Harman reasoned that enzymatic reactions using molecular oxygen create, on occasion, O2 radicals. Harman hypothesized that lower levels of oxygen free radicals, by reducing enzymatic activities that utilized molecular oxygen (O2), would result in increased longevity.

Mitochondria are the primary sites of O2 consumption within the cell, suggesting that mitochondria are also the primary sites of oxygen-free radical production. Therefore, according to the free radical theory of aging, reduced mitochondrial function should correlate with increased longevity. Indeed, yeast lacking mitochon-drial DNA, thereby having to survive on metabolic fermentation, have increased replicative life spans compared to wild-type yeast cells (Jazwinski 1990). Furthermore, long-lived, calorie-restricted mice show reduced expression of several key metabolic genes, including several mitochondrial genes (Lee et al. 1999). Taken together, these studies suggest that reduced mitochondrial function should increase longevity of multicellular organisms.

Research in nematodes identified the mitochondrial electron transport and ATP synthase as regulators of the aging process. Three studies demonstrated that reduc tion of function of several mitochondrial genes extends the life span of fully developed, adult worms. Siegfried Hekimi's lab showed that a single mutation in an iron sulfur component of Complex III, isp-1, increased longevity. This mutation decreases oxygen consumption, suggesting that it lowers the activity of the electron transport chain (Feng 2001). isp-1 mutant worms display delayed development and reduced rates of other physiological processes, including eating, movement, and defecation (Feng 2001). Two independent RNAi-based screens also showed that components of the mitochondrial electron transport chain increased longevity when they were inactivated using RNAi (Dillin et al. 2002, Lee et al. 2003). It was found that RNAi inactivation of components of Complex I, III, IV, and the ATP synthase increases longevity of wild-type worms (Dillin et al. 2002). These results are consistent with those determined using an RNAi-based screen in Gary Ruvkun's lab (Lee et al. 2003). Similar to the properties of isp-1 mutant worms, RNAi of the mitochondrial electron transport chain genes reduced ATP levels, O2 consumption and slowed the rate of development and other physiological processes, including eating, movement and defecation (Dillin et al. 2002, Lee et al. 2003).

Mutations in another worm gene, clk-1, lengthens life span (Wong et al. 1995) but does not appear to reduce respiration (Braeckman et al. 1999, Miyadera et al. 2001). clk-1 mutations prevent the synthesis of ubiquinone (Miyadera et al. 2001), which is required for respiratory chain activity. clk-1 mutants are viable because they acquire ubiquinone from the bacteria in their culture media (Jonassen et al. 2001).

How does mitochondrial activity affect life span in C. elegans? One possibility is that mitochondrial activity inhibits the insulin/IGF-1 signaling pathway (Guarente 2000). Reducing the activity of DAF-2, an insulin/IGF-1 receptor homolog, or downstream signaling components, extends life span approximately two-fold. This life span extension requires activity of the forkhead-family transcription factor DAF-16. However, isp-1, clk-1 mutations or RNAi of respiratory chain components extend the life span of daf-16 mutations (Dillin et al. 2002, Feng 2001, Lee et al. 2003, Wong et al. 1995). In addition, the already long life span of daf-2(e1370) mutants is further extended by isp-1, clk-1 mutations, or RNAi of respiratory chain components. Moreover, unlike reduction of respiratory chain activity, reduction of insulin/IGF-1 signaling is known to cause a significant increase in ATP levels (Braeckman et al. 1999, Dillin et al. 2002). Finally, both daf-2 and daf-16 act exclusively in adults to regulate life span (Dillin et al. 2002). Together these findings indicate that respiratory-chain RNAi does not increase life span by inhibiting the DAF-2 pathway.

Not all mitochondrial ETC lesions increase longevity. The mev-1(kn1) mutation is probably the best example of a mutation that decreases mitochondrial activity, but does not increase longevity. The mev-1(kn1) mutation was identified in a genetic screen to identify mutations that resulted in worms that were more sensitive to the drug methyl viologen (Paraquat). Cells treated with Paraquat produce excess oxygen-free radicals. mev-1(kn1) mutant animals are hypersensitive to Paraquat, are short lived, and have reduced mitochondrial respiratory rates (Hosokawa et al. 1994, Ishii et al. 1998, Ishii et al. 1990). Additionally, mev-1(kn1) mutant animals have higher levels of oxygen-free radicals compared to wild-type animals (Ishii et al. 1998, Senoo-Matsuda et al. 2001). mev-1 encodes the cytochrome b large subunit of Complex II (Ishii et al. 1998). Similar to mev-l(knl) mutant animals, gas-1(fc21) mutant animals are also hypersensitive to Paraquat, short lived, have reduced ETC activity, and have higher levels of oxygen-free radicals compared to wild-type animals (Senoo-Matsuda et al. 2001). gas-1 encodes the 49 kilo Dalton iron-containing subunit of mitochondrial electron transport chain complex I (Kayser et al. 1999). Besides mev-1 and gas-1 mutations, nuo-1(ua1) and atp-2(ua2) mutant worms also have reduced mitochondrial respiratory function (Tsang et al. 2001). nuo-1(ua1) and atp-2(ua2) mutant animals arrest during development and do not grow to reproductive adulthood (Tsang et al. 2001). nuo-1 encodes the NADH- and FMN-binding subunit of complex I and atp-2 encodes an active site subunit of complex V, the ATP synthase.

Taken together, a clear correlation between metabolic activity and longevity cannot be derived from these studies. For example, clk-1 mutant animals are long-lived, but have normal respiratory rates, and RNAi of several ETC components results in increased longevity and decreased metabolic rates. Therefore, defining the role that mitochondria play in the aging process will be essential to elucidating the correlation between metabolic rates and longevity, as originally proposed by Pearl in the 1930s.

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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