IGF-1 mediated phosphorylation of specific IRS-1 serines in Ames dwarf fibroblasts is associated with longevity.

Insulin/IGF-1 signaling involves phosphorylation/dephosphorylation of serine/threonine or tyrosine residues of the insulin receptor substrate (IRS) proteins and is associated with hormonal control of longevity determination of certain long-lived mice. The stimulation of serine phosphorylations by IGF-1 suggests there is insulin/IGF-1 crosstalk that involves the phosphorylation of the same serine residues. By this mechanism, insulin and IGF-1 mediated phosphorylation of specific IRS-1 serines could play a role in longevity determination.We used fibroblasts from WT and Ames dwarf mice to examine whether: (a) IGF-1 stimulates phosphorylation of IRS-1 serines targeted by insulin; (b) the levels of serine phosphorylation differ in WT vs. Ames fibroblasts; and (c) aging affects the levels of these serine phosphorylations which are altered in the Ames dwarf mutant. We have shown that IRS-1 is a substrate for IGF-1 induced phosphorylation of Ser307, Ser612, Ser636/639, and Ser1101; that the levels of phosphorylation of these serines are significantly lower in Ames vs. WT cells; that IGF-1 mediated phosphorylation of these serines increases with age in WT cells. We propose that insulin/IGF-1 cross talk and level of phosphorylation of specific IRS-1 serines may promote the Ames dwarf longevity phenotype.

Dermal fibroblasts from long-lived Ames dwarf mice maintain their in vivo resistance to mitochondrial generated reactive oxygen species (ROS).

Activation of p38 MAPK by ROS involves dissociation of an inactive, reduced thioredoxin-ASK1 complex [(SH)(2)Trx-ASK1]. Release of ASK1 activates its kinase activity thus stimulating the p38 MAPK pathway. The level of p38 MAPK activity is, therefore, regulated by the balance of free vs. bound ASK1. Longevity of Ames dwarf mice is attributed to their resistance to oxidative stress. The levels of (SH)(2) Trx-ASK1 are more abundant in young and old dwarf mice compared to their age-matched controls suggesting that the levels of this complex may play a role in their resistance to oxidative stress. In these studies we demonstrate that dermal fibroblasts from these long-lived mice exhibit (a) higher levels of (SH)(2)Trx-ASK1 that correlate with their resistance to ROS generated by inhibitors of electron transport chain complexes CI (rotenone), CII (3-nitropropionic acid), CIII, (antimycin A), and H(2)O(2)-mediated activation of p38 MAPK, and (b) maintain their in vivo resistance to ROS generated by 3NPA. We propose that elevated levels of (SH)(2)Trx-ASK1 play a role in conferring resistance to mitochondrial generated oxidative stress and decreased endogenous ROS which are characteristics of longevity determination.

Thioredoxin-ASK1 complex levels regulate ROS-mediated p38 MAPK pathway activity in livers of aged and long-lived Snell dwarf mice.

We have proposed that the age-associated increase of reactive oxygen species (ROS) by electron transport chain (ETC) dysfunction may cause the elevated basal level of p38 MAPK stress response pathway activity. However, the mechanism by which ROS activates this pathway is not clear. Here we propose that activation of the p38 MAPK pathway by complex I (CI) generated ROS, in response to rotenone (ROT) treatment, is based on the ability of reduced Trx to bind to and inhibit ASK 1 and its release from the complex upon oxidation. This balance of free vs. bound ASK1 regulates the level of p38 MAPK pathway activity. To support this mechanism we demonstrate that the production of ROS by ROT treated AML12 hepatocyte cells dissociates the Trx-ASK1 complex, thereby increasing p38 MAPK pathway activity. This mechanism is supported by the ability of N-acetyl cysteine (NAC) to prevent dissociation of Trx-ASK1 and activation of the p38 MAPK pathway. We also demonstrated that the ratio of ASK1/Trx-ASK1 increases in aged mouse livers and that this correlates with the increased basal activity of the p38 MAPK pathway. The longevity of Snell dwarf mice has been attributed to their resistance to oxidative stress. A comparison of the levels of Trx-ASK1 in young and aged dwarfs showed a higher abundance of the complex than in their age-matched controls. These results, which are indicative of a decreased level of oxidative stress, suggest that increased ROS production in aged liver may alter the ratio of ASK1 and Trx-ASK1, thereby increasing the age-associated basal level of p38 MAPK pathway activity.

Akt/PKB and p38 MAPK signaling, translational initiation and longevity in Snell dwarf mouse livers.

The insulin/IGF-1/GH and p38 MAPK signaling pathways play a key role in the regulation of protein synthesis. The regulation of GH and TSH secretion hormones, that affect the activity of these pathways, plays an important role in the decline of rates of protein synthesis in aged rodent tissues. Studies have indicated that longevity of the Snell dwarf (Pit-1) mouse mutant is associated with the reduction of function of the insulin/IGF-1/GH signaling pathway. We have previously shown that PI3K activity, a signaling protein that plays a key role in the regulation of translation, is also dramatically decreased in the Snell dwarf liver suggesting that the protein synthesis-signaling pathway may be attenuated in this long-lived mouse. Similarly, signaling via p38 MAPK also plays a role in the regulation of protein synthesis. In this study we examined the activities of these signaling pathways to determine if the translation-signaling pathway is altered in young versus aged Snell dwarf mouse livers. Our data indicate that the phosphorylation and kinase activities of Akt/PKB and p38 MAPK, and the levels of phosphorylation of downstream regulators of translation are decreased in dwarf mouse livers. Thus, the overall activities of major components of the translational initiation pathway are decreased in the long-lived Snell dwarf mouse livers. We propose that down-regulation of protein synthesis may be an important characteristic of the Pit-1 mutation and longevity of the Snell dwarf mouse.

Altered oxidative stress response of the long-lived Snell dwarf mouse.

Several single gene mutations in mice that increase the murine life span have been identified, including the Pit-1 mutation which results in the Snell dwarf (Pit1(dw/dw)), however, the biological mechanism of this life-span extension is still unclear. Based on studies that show oxidative stress plays an important role in the aging process, we hypothesized that the increased longevity seen in Snell dwarf mice may result from a resistance to oxidative stress. We report that Snell dwarf mice respond to oxidative stress induced by 3-NPA differently than their wild type littermates. This altered response results in diminished activation of the MEK-ERK kinase cascade and virtually no phosphorylation of c-Jun at Ser63 in dwarf mice after 3-NPA treatment, despite a robust phosphorylation of Ser63 in wild type mice. We propose that this altered management of oxidative stress in dwarf mice is partially responsible for the increased longevity in Snell dwarf mice.

Age-associated changes in SAPK/JNK and p38 MAPK signaling in response to the generation of ROS by 3-nitropropionic acid.

Mitochondrial dysfunction has been identified as a major source of oxidative stress in aged tissues. In this study we asked whether activities of components of the SAPK/JNK and p38 MAPK stress response signaling pathways are indicative of oxidative stress in aged mouse livers and whether these pathways are responsive to oxidative stress generated by 3-nitropropionic acid (3-NPA), an inhibitor of complex II (succinic dehydrogenase). We asked whether (a) aging affects the basal activity of the SAPK/JNK stress signaling pathway; (b) specific isoforms of JNK, i.e. 46 or 54 kDa JNKs are activated by 3-NPA; (c) aging affects the response of this signaling pathway to 3-NPA; (d) there is a cross pathway activation of JNK or p38 MAPK by upstream activators. Our studies have shown that although their protein pool levels are not altered, the basal JNK activities using c-Jun as substrate is elevated. Furthermore, in aged livers, JNK activity is induced to a greater extent and takes longer to recover from 3-NPA treatment. The activities of the upstream activators of JNKs, MAP kinase kinase (MKK) 4 and 7, are also elevated in livers of aged C57BL/6 male mice. These activator kinases, which are induced (phosphorylated) by 3-NPA in young livers, are not inducible by this inhibitor in aged livers. In fact, these proteins are highly phosphorylated in the control aged livers and are dephosphorylated in response to 3-NPA. Finally, we demonstrate for the first time that MKK7 serves as an upstream activator of p38 MAPK and that MKK3 and MKK6 activates 54 kDa JNK2 in aged liver. Our studies suggest that failure to respond to 3-NPA may be indicative of the susceptibility of aged tissue to oxidative stress, supporting our hypothesis that aged tissues (especially liver) develop a state of chronic stress even in the absence of a challenge.

The effect of aging on p38 signaling pathway activity in the mouse liver and in response to ROS generated by 3-nitropropionic acid.

Since mitochondrial dysfunction is a major source of oxidative stress in aged tissues, we asked whether the basal activities of stress response signaling pathway(s) are indicative of oxidative stress in aged tissues. To address this issue we asked whether: (a). aging affects the basal activity of the p38 MAPK stress signaling pathway; (b). the p38 MAPK pathway is activated by 3-nitropropionic acid (3-NPA), an inhibitor of complex II (succinic dehydrogenase) and generator of reactive oxygen species (ROS); (c). aging affects the response of the p38 alpha signaling pathway to 3-NPA. Our studies have shown that the basal kinase activities of p38 alpha, its upstream activator, MKK3, and its downstream substrate, ATF-2, are elevated in livers of aged C57BL/6 male mice and that these kinase activities, which are induced by 3-NPA in young livers, do not occur in aged livers. Furthermore, although aging does not affect their protein pool levels there are specific increases in phosphorylation of threonine residues in the p38 alpha and ATF-2 catalytic sites that might account for the increased basal level kinase activities in the aged livers. Our studies suggest that failure to respond to 3-NPA may be a factor in the susceptibility of aged tissue to oxidative damage, and support our hypothesis that aged tissues (especially liver) develop a state of chronic stress even in the absence of a challenge.

Implications for the insulin signaling pathway in Snell dwarf mouse longevity: a similarity with the C. elegans longevity paradigm.

Mutation analyses in the nematode, Caenorhabditis elegans, and mice have identified genes that increase their life-span via hormonal signal transduction, i.e. the insulin/insulin-like growth factor-1 (IGF-1) pathway in nematodes, and the growth hormone (GH)-thyriod stimulating hormone (TSH)-prolactin system in Snell dwarf mouse mutants. We have shown that the GH deficiency due to Pit1 mutation in the long-lived Snell dwarf mice may decrease circulating insulin levels, thereby resulting in a decreased activity of the insulin/IGF-1 signaling pathway. The data presented are consistent with our hypothesis that the decreased circulating insulin levels resulting from the Pit1 mutation mimics a physiological state similar to that proposed to occur in the long-lived C. elegans, daf-2 mutant. Our studies demonstrate a series of changes in components of the insulin/IGF-1-signaling pathway that suggest a reduction-of-function of this pathway in the aged dwarf. These include a decreased IRS-2 pool level, a decrease in PI3K activity and its association with IRS-2 and decreased docking of p85alpha to IRS-2. Our data also suggest a preferential docking of IRS-2-p85 alpha -p110 alpha in the aged dwarf liver and IRS-2-p85 alpha -p110 beta in the aged control. We speculate that the preference for the p110 alpha-containing complex may be a specific characteristic of a downstream segment of the longevity-signaling cascade. We conclude that the Pit1 mutation may result in physiological homeostasis that favors longevity, and that the Snell dwarf mutant conforms to the nematode longevity paradigm.

Effects of the Pit1 mutation on the insulin signaling pathway: implications on the longevity of the long-lived Snell dwarf mouse.

Mutations in Caenorhabditis elegans and mice have identified candidate genes that increase their lifespan via hormonal signal transduction, i.e. the insulin/IGF-1-like pathway. In this study we propose that longevity of the Snell dwarf (Pit1(dw)/Pit1(dw)) mouse is associated with a decrease of the insulin/IGF-1 signaling pathway caused by the Pit1 mutation. We recently demonstrated that the growth hormone deficiency of the dwarf mouse alters circulating insulin levels, thereby resulting in a decreased activity of the insulin/IGF-1 signaling pathway, which is a determining factor in the increased nematode lifespan. The decreased activity of the insulin/IGF-1 signaling pathway is indicated by decrease of (a) IRS-two pool levels; (b) docking of p85 alpha to IRS-2; (c) docking of p 85 alpha to p110 alpha or p110 beta, and (d) IRS-2-associated PI3K activity. In this study we present data suggesting that the InR beta-IRS-1-PI3K pathway is attenuated in the Snell dwarf mouse liver. Our data show that the PI3K activity associated with IRS-1, the docking of IRS-1 to InR beta and the docking of p85 alpha to IRS-1 are attenuated in the aged Snell dwarf. Our studies suggest that the Pit1 mutation results in a decreased activity of the insulin/IGF-1 pathway; that this plays a key role in the longevity of the Snell dwarf mouse and conforms to the nematode longevity paradigm.