Sci. Aging Knowl. Environ., 9 June 2004
Vol. 2004, Issue 23, p. pe25
[DOI: 10.1126/sageke.2004.23.pe25]

PERSPECTIVES

Inside Insulin Signaling, Communication Is Key to Long Life

Adam Antebi

Adam Antebi is in the Max-Planck-Institut für Molekulare Genetik, Ihnestrasse 73, D-14195 Berlin, Germany. E-mail: antebi{at}molgen.mpg.de

http://sageke.sciencemag.org/cgi/content/full/2004/23/pe25

Key Words: insulin/IGF signaling • fat body • Drosophila • oxidative stress

One of the startling themes to emerge from studies of aging in model systems is that longevity is regulated and under genetic control (see "Aging Research Grows Up"). Remarkably, some of the signaling pathways that have been shown to influence organismal life span are evolutionarily conserved (2) (see "One for All"). As first demonstrated in the nematode Caenorhabditis elegans, increased insulin/insulin-like growth factor (IGF) signaling is proaging, whereas decreased signaling promotes longevity, resulting in a doubling of adult life span (3, 4) (see Johnson Review). Similarly, mutations in the fly gene chico, which encodes an insulin-related receptor substrate (5), or heteroallelic combinations of the fly insulin/IGF receptor (6) (InR) extend life by 36 to 85%. In the mouse, +/– heterozygotes of IGF-1 receptor knockouts live 33% longer than wild-type mice (7). In many cases, these animals live extended, healthier lives, with relatively little cost to fertility and metabolism. In all three systems, it is a modest inhibition of the pathway that prolongs life. Even more ancient remnants of this circuit exist in yeast, which obviously lacks insulin/IGF signaling per se (for example, the AKT kinase homolog SCH9) (8). In this Perspective, I discuss results presented in a recent Nature paper (1) that show that suppression of insulin/IGF signaling specifically in the adipose tissue of the fly Drosophila melanogaster retards organismal aging by a cell-non-autonomous mechanism.

The extraordinary finding that single-gene mutations act in a conserved molecular pathway to extend life has fundamentally shifted the prevalent view of aging. Instead of idiosyncratic mechanisms, ancient strategies must have evolved to retard aging under duress, presumably in order to outlast adversity until conditions suitable for reproduction arose (see "Hard Times Teach Life-Extending Lessons"). Thus, findings in these simple genetic models may be broadly applicable to higher organisms. But how far do the similarities hold in species with such diverse life histories, body plans, and physiology? The answer is, surprisingly far (see "Growing Old Together").

First, work from various quarters suggests that insulin-like signaling exists in a variety of species and operates by a similar mechanism. In response to insulin-like peptides, the insulin/IGF receptor (called DAF-2 in worms and InR in flies) stimulates a conserved kinase cascade that ultimately leads to the phosphorylation of a FOXO forkhead transcription factor (called DAF-16 in worms; dFOXO in flies; and FOXO1, FOXO3a, and FOXO4 in mammals). Phosphorylated FOXO remains in the cell's cytoplasm, which produces proaging phenotypes. In contrast, when insulin/IGF signaling is blocked, FOXO is not phosphorylated; thus it enters the nucleus and regulates the transcription of genes that enhance longevity (for example, see Larsen Perspective). These findings reveal that FOXO proteins constitute central transcriptional mediators of longevity that are normally inhibited by active insulin/IGF signaling.

External stimuli that activate FOXO transcription factors include heat and oxidative stress, ultraviolet radiation, and nutrient limitation (9-12). FOXO target genes include those that encode proteins that manage these cellular affronts (13-17), suggesting that FOXO senses and then mitigates cellular stresses. Thus, aside from its traditional role in growth and metabolism, insulin/IGF signaling, when repressed, evokes a generalized stress response. In fact, resistance to oxidative challenge correlates most tightly with longevity, and somatic endurance may underlie organismal long life. The oxidative damage theory of aging posits that senescence arises from injury to cellular components inflicted by reactive oxygen radicals (see "The Two Faces of Oxygen"). It is likely that decreased insulin/IGF signaling minimizes damage to and increases repair of cellular macromolecules, all of which supports this major theory of aging. However, genes involved in metabolism, immune protection, and various unknown functions may also contribute to longevity by other means (13, 14). Moreover, dietary restriction, which increases longevity in diverse species (see "Monkey in the Middle"), might deploy insulin/IGF signaling, although this hypothesis remains to be proven.

But when and where is the inhibition of insulin/IGF signaling required to achieve enhanced longevity? Must FOXO transcription factors be active throughout development or only in the adult? Must they be expressed in all cells or only some? Although the answers to these seemingly simple questions are turning out to be surprisingly complex, excitement in the field is mounting, because common themes are emerging across the various genetic models. In the nematode, activation of DAF-16 in the adult is sufficient to extend life span (18) (see Sonntag and Ramsey Perspective), suggesting that developmental contributions toward longevity are minimal. This is important, because inhibition of the pathway during larval life can arrest development and cause numerous deleterious effects. Endogenous DAF-16 is widely expressed, but selective activation, primarily in the intestine and secondarily in neural tissue, promotes longer life (19-21) (see "Visceral Reaction"). Mosaic analysis of DAF-16 suggests that other tissues also contribute significantly to life span determination (19), but they remain to be clearly identified. That expression only in selective tissues can slow aging of the entire organism is surprising, because it implies that specific tissues dictate long life, perhaps through secondary hormonal signals. But are these features peculiar to the worm?

According to a recent paper (1), apparently not. Another layer of conservation may lie at the level of how specific tissues communicate to establish organismal longevity. In a paper published this week in Nature, Tatar and colleagues explore the time and tissue focus of dFOXO in the regulation of fly life span. Because ubiquitous constitutive expression of dFOXO during development was lethal, the authors used a genetic trick to precisely regulate its activity. First, a version of dFOXO that was independent of its normal kinase control was generated by mutating the dfoxo gene so that the target phosphorylation sites in the dFOXO protein were converted to alanine. Then the mutated dfoxo gene was fused to nucleic acid sequences that encode the ligand-binding domain of the progesterone receptor (PR), rendering dFOXO nuclear localization dependent on exogenously applied mifepristone, a PR ligand. Thus, in the absence of mifepristone, dFOXO remains in the cytoplasm, and its activity is effectively blocked; when flies are fed mifepristone, dFOXO translocates to the nucleus and regulates target genes. Finally, by placing dFOXO expression under the control of tissue-specific inducible systems, the authors were able to conditionally activate dFOXO in a particular tissue at a particular time and thus bypass developmental complications and lethality.

First, Tatar and colleagues found that activating the engineered version of dFOXO by feeding with mifepristone in the adult fly indeed extended life span, thus revealing that longevity results not from the imprint of developmental events, but from gene regulation that occurs in adult animals. Long life was observed, however, only when dFOXO was activated in select tissues. In particular, expression within the head fat body, but not the abdominal fat body, increased life span by 13 to 56%. Similarly, overexpression of PTEN, a phosphatase that activates the endogenous dFOXO, extended life when expressed in the same tissue. Pan-neural or glial cell expression of dFOXO had little effect on life span, whereas expression in the neurolemma, a neuronal support tissue, resulted in a slight increase, suggesting modest neural contributions. These manipulations also increased resistance to oxidative challenge but had little effect on fertility, further demonstrating that stress resistance, not reproductive status, correlates with longevity.

Remarkably, dFOXO activation in the head fat body not only increased fat deposition in this tissue but increased fat deposition in the peripheral fat body as well, suggesting that dFOXO has both cell-intrinsic (autonomous) and hormonal (non-autonomous) effects. The fat bodies in flies are analogous to the worm intestine and vertebrate adipose tissue and liver--sites of fat deposition, metabolic control, and endocrine signaling. The respective roles of head and abdominal fat bodies are poorly understood, but the work described here suggests unique and unexpected functions. Significantly, a tissue-specific knockout of the mouse insulin receptor in adipose tissue also results in a modest 18% increase in life span (22) (see "Lasting Without Fasting"). By inference, adipose tissue too can produce signals that influence vertebrate longevity.

So what might be the nature of these non-autonomous hormonal signals? An important clue came when Tatar and colleagues examined endogenous dFOXO in peripheral tissues. Activation of dFOXO in the head fat body resulted in the nuclear localization of endogenous dFOXO in the abdominal fat body. This suggests that dFOXO inhibits insulin signaling itself by a hormonal mechanism. Consistent with this observation, specific down-regulation of the Drosophila insulin-like peptide (ILP) dILP2 within neurosecretory cells was also observed. Finally, ablation of dILP2-expressing cells increased fly life span (23), suggesting that blunted dILP2 expression could account for systemic control of longevity, although this hypothesis needs to be tested directly. Moreover, it will be interesting to specifically examine the behavior of FOXO within insulin producing cells.

Similarly, in the worm an insulin relay was previously suggested. First, various ILP genes were shown to be targets of DAF-16 down-regulation in gene expression profile experiments (13). Second, when DAF-16 was overexpressed in the intestine, induction of a DAF-16 target, the manganese-dependent superoxide dismutase gene sod-3, was observed not only in the intestine but in the epidermis and muscle as well, revealing autonomous and non-autonomous action (19). However, distal expression of sod-3 was dependent on the presence of DAF-2/insulin/IGF receptor and DAF-16 within these peripheral tissues, revealing that this non-autonomy demands that insulin/IGF signaling be intact. It remains to be seen whether intestinal ILPs or some other molecules mediate these effects in C. elegans.

A simple model to explain all of these results is that when FOXO activity is inhibited in signaling tissues, the secreted insulin agonists stimulate the insulin/IGF pathway in responding tissues, propagating the signal throughout the body. Conversely, tissue-specific activation of FOXO may stymie the spread of a systemic agonist or promote expression of an antagonist of the insulin/IGF pathway. In the worm, ILPs are expressed in various tissues, including the intestine, nervous system, muscle, and epidermis (24); and, in principle, these tissues can variably communicate through insulin/IGF signaling (19). However, this model raises a paradox in the fly. None of the seven identified Drosophila ILPs are expressed in the head fat body, so how is tissue communication achieved? One possibility is that the fat body could produce insulin-like binding proteins that regulate the transport or half-life of circulating dILPs. In agreement with this hypothesis, nutrient deprivation restricts the expression of the Drosophila acid labile subunit (ALS), a component of ternary insulin-like binding complexes, within the larval fat body (25). Alternatively, FOXO could regulate other hormones or metabolites that impinge on dILP synthesis in the insulin-producing cells. Indeed, insulin signaling regulates the production of fly juvenile hormone and ecdysone (6, 26) (see Zwaan Perspective), but it is perfectly possible that these hormones also impact insulin signaling.

In the worm, other hormonal pathways that are not mediated by insulin/IGF also likely exist downstream of DAF-16. Otherwise, it is difficult to explain the results of experiments in which activated DAF-16 in the intestine increases organismal survival, despite the lack of the DAF-2/insulin/IGF receptor and DAF-16 in distal receiving tissues (19). Accordingly, another major class of genes regulated by C. elegans FOXO includes those involved in the metabolism of lipophilic hormones (13). Moreover, insulin/IGF signaling has been shown to converge on lipophilic hormone signaling in the regulation of developmental arrest at the dauer diapause, an alternate long-lived third larval stage (27, 28). In the future, it will be fascinating to correlate the gene expression dynamics with phenotypes of the diverse insulin-like ligands as well as other hormones in response to tissue-specific genetic manipulations.

Finally, why should such complicated tissue signaling be employed? By polling the hormonal status from a variety of tissues, the organism could evaluate the various levels of organ stress and, accordingly, come to some type of organismal consensus (Fig. 1). If the general consensus is overall insulin release, then pro-reproductive, pro-aging modes would be selected. If the overall view is insulin suppression, then the organism would select antiaging programs of somatic endurance to survive adversity. Moreover, such a mechanism could allow coordination of the various tissues as well as fine-tuned and graded responses. Although seemingly resembling a democratic process, clearly some tissues have weighted votes. Naturally, reproductive tissues, such as the gonad and germline, must have strong input. Indeed, signals from the C. elegans germline inhibit nuclear localization of intestinal DAF-16 (10). Also, ablation of the germline increases organismal life span in a manner dependent on FOXO as well as on the DAF-12 nuclear hormone receptor pathway (29, 30). Moreover, adipose tissue is a strong indicator of the metabolic reserves of the organism and a good predictor of reproductive success. Finally, the nervous system detects environmental signals, which are also important indicators of nutrient availability. Non-autonomous signaling fosters proper feedback among the tissues, while autonomous action regulates the nuts-and-bolts machinery of reproduction, soma maintenance, macromolecule repair, and metabolism. Integration of the two types of regulation ensures maximal survival and reproductive success.



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Fig. 1. A general model of organismal aging regulated by intercellular insulin/IGF signaling. In the pro-aging reproductive mode, unidentified non-autonomous signals from the germline inhibit adipose FOXO, which conceivably permits expression of ILPs and/or other endocrine regulators (steroids?) from adipose tissue. This can also be accomplished by activating signaling through the insulin receptor (InR). It is possible that activation of insulin/IGF signaling (InR) in insulin producing cells (ipcs) blocks FOXO activity and promotes expression of ILPs that feed back (dotted lines) on endocrine tissues to reinforce reproductive status and turn on reproductive target genes in peripheral tissues (right-pointing open arrowheads). In the antiaging mode, absent hormonal signals activate FOXO, which suppresses the spread of insulin-like agonists (ILPs) from adipose and/or other insulin-producing tissues. Target genes that promote survival are expressed in endocrine and peripheral tissues (left-pointing open arrowheads).

 
That FOXO works by both cell-autonomous and -non-autonomous mechanisms to regulate longevity, stress resistance, fat deposition, and insulin signaling in both worms and flies indicates the remarkable conservation of this signaling network. Moreover, while the origin of such hormonal signals resides primarily in adipose tissue, other tissues likely also contribute, because the full complement of longevity seen in insulin/IGF signaling mutants is not entirely recapitulated by selective expression of FOXO. As mentioned above, blunted insulin signaling in adipose tissue modestly increases murine life span, and it will be particularly exciting to see whether some of the tissue-specific non-autonomous mechanisms that regulate life span in worms and flies also hold true in mammals.


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Citation: A. Antebi, Inside Insulin Signaling, Communication Is Key to Long Life. Sci. Aging Knowl. Environ. 2004 (23), pe25 (2004).








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