insulin/igf-like signalling, the central nervous system ... › 1123 › e0107fcd4fc9... ·...

Biochem. J. (2009) 418, 1–12 (Printed in Great Britain) doi:10.1042/BJ20082102 1

REVIEW ARTICLEInsulin/IGF-like signalling, the central nervous system and agingSusan BROUGHTON and Linda PARTRIDGE1

UCL Institute of Healthy Aging, GEE (Genetics, Evolution and Environment), University College London, Gower St., London WC1E 6BT, U.K.

Enormous strides in understanding aging have come from thediscovery that mutations in single genes can extend healthy life-span in laboratory model organisms such as the yeast Saccharo-myces, the fruit fly Drosophila melanogaster, the nematode wormCaenorhabditis elegans and the mouse. IIS [insulin/IGF (insulin-like growth factor)-like signalling] stands out as an important,evolutionarily conserved pathway involved in the determinationof lifespan. The pathway has diverse functions in multicellularorganisms, and mutations in IIS can affect growth, development,metabolic homoeostasis, fecundity and stress resistance, as wellas lifespan. The pleiotropic nature of the pathway and the oftennegative effects of its disruption mean that the extent, tissueand timing of IIS manipulations are determinants of a positiveeffect on lifespan. One tissue of particular importance for lifespanextension in diverse organisms is the CNS (central nervoussystem). Although lowered IIS in the CNS can extend lifespan, IIS

is also widely recognized as being neuroprotective and importantfor growth and survival of neurons. In the present review, wediscuss our current understanding of the role of the nervous systemin extension of lifespan by altered IIS, and the role of IIS indetermination of neuronal function during aging. The nervoussystem can play both endocrine and cell-autonomous roles inextension of lifespan by IIS, and the effects of IIS on lifespanand neuronal function can be uncoupled to some extent. Tissue-specific manipulation of IIS and the cellular defence mechanismsthat it regulates will better define the ways in which IIS affectsneuronal and whole-organism function during aging.

Key words: aging, central nervous system, health, insulin/IGF(insulin-like growth factor)-like signalling, lifespan, neuronalsurvival.

INTRODUCTION

The IIS [insulin/IGF (insulin-like growth factor)-like signalling]pathway is ubiquitous in multicellular animals and probablyplayed a central role in the evolution of multicellularity itself[1]. The pathway has diverse functions, and mutations that alterIIS can have pleiotropic effects on growth [2–5], development[6], metabolic homoeostasis [7] and fecundity [8–10]. Althoughalterations in IIS can have severely detrimental effects, an obviousone being diabetes in mammals, lowered IIS has emerged as animportant, evolutionarily conserved means of extending lifespanin the nematode worm Caenorhabditis elegans, the fruit flyDrosophila melanogaster and the mouse Mus musculus [11–15].

Improvements in public health and lifestyle in many developedcountries have resulted in increasing human life expectancy[16]. Despite the increases in individual health that underlie thistrend, more people are hence living long enough to experienceaging-related disease and loss-of-function. It is thus important toevaluate the health and function of long-lived model organisms asthey age, to determine whether interventions that extend lifespanalso have the potential to delay or attenuate aging-related diseaseand functional senescence. Lowered IIS can indeed improvehealth at older ages. For instance, long-lived mice that lackIRS [IR (insulin receptor) substrate]-1, despite their mild insulinresistance when young, maintain glucose homoeostasis better thancontrols at older ages. They also show improvements in immune

profile and motor performance, as well as lowered incidenceof osteoporosis, cataract and ulcerative dermatitis [17]. Long-lived invertebrate model organisms show similar improvementsin health. Drosophila lacking chico, the single fly IRS, show in-creased lifespan together with improved immune function [18]and a slower decline in the one measure of behavioural health, neg-ative geotaxis, so far examined [19]. Long-lived C. elegans withmutations in age-1 (AGEing alteration-1), the gene encoding thecatalytic subunit of the worm PI3K (phosphoinositide 3-kinase),or daf-2 (abnormal DAuer Formation-2), the gene encodingthe worm insulin/IGF-1 receptor, show improved thermotaxis-associative conditioning behaviour [20] and enhanced immunity[21] with age. Inhibition of the IIS pathway in worms inhibitstumour growth [22,23] and reduces the toxicity of aggregrate-prone proteins in a worm model of Alzheimer’s disease [24], andin humans the pathway is being targeted as a therapy for cancer[25]. Thus inhibition of specific IIS components can improvebroad aspects of health in a range of model organisms and hasthe potential to attenuate functional senescence and age-relateddisease in humans.

The signalling mechanisms, biochemical changes and thetissues that mediate the effects of lowered IIS on lifespan, healthand function are starting to be elucidated. Studies using systemsthat allow temporal and spatial control of IIS manipulation, such asfeeding-induced RNAi (RNA interference) in the worm, RU486-induced GAL4/UAS (upstream activation sequence) in the fly

Abbreviations used: CNS, central nervous system; DILP, Drosophila insulin-like peptide; FOXO, forkhead box O; dFOXO, Drosophila FOXO; GH, growthhormone; HSF, heat-shock factor; IGF, insulin-like growth factor; IGF-1R, IGF-1 receptor; IIS, insulin/IGF-like signalling; INS, insulin-like peptides; IR, insulinreceptor; IRS, IR substrate; JH, juvenile hormone; JNK, c-Jun N-terminal kinase; mNSC, median neurosecretory cell; NPY, neuropeptide Y; Nrf2, nuclearfactor-erythroid 2 p45 subunit-related factor 2; PDK, phosphoinositide-dependent kinase; PI3K, phosphoinositide 3-kinase; PKB, protein kinase B; PTEN,phosphatase and tensin homologue deleted on chromosome 10; dPTEN, Drosophila PTEN; SIRT, sirtuin; sNPF, short neuropeptide F; TOR, target ofrapamycin.

1 To whom correspondence should be addressed (email [email protected]).

c© The Authors Journal compilation c© 2009 Biochemical Society

2 S. Broughton and L. Partridge

[26] and the Cre/lox system in the mouse [27], have begunto refine the analysis, and it is becoming clear that specificalterations of IIS in specific tissues and at specific times areimportant determinants of the effect on lifespan of reduced IISactivity. Although not yet investigated in mammals, lowered IIS ininvertebrate model organisms during the early adult period hasbeen found to be sufficient for lifespan extension, and to have noeffect on reproductive capacity [10,28–32]. It is thus possible toextend lifespan while avoiding one major detrimental pleiotropiceffect, at least in invertebrates.

Tissue-specific manipulation of gene expression has identifiedsome of the evolutionarily conserved sites that play a role inextension of lifespan by IIS [10,30,31,33–36]. Fat and neuronaltissues appear to be particularly important. Fat-specific knockoutof the IR in mice [35] and overexpression of dPTEN [DrosophilaPTEN (phosphatase and tensin homologue deleted on chromo-some 10)] or dFOXO [Drosophila FOXO (forkhead box O)] inthe fat body of flies [30,31] can extend lifespan. In worms, theintestine, which serves as a fat storage organ, similarly plays aprominent role [34]. Brain-specific knockout of the IRS-2 in miceextends lifespan [36], as does ablation of mNSCs (median neuro-secretory cells) that secrete insulin-like peptides in the brain offlies [10]. Neuronal tissue has been implicated in the extensionof lifespan by lowered IIS in several studies in worms [33,34,37].

The discovery that the CNS (central nervous system) is involvedis perhaps not surprising given its role in co-ordination ofphysiology and behaviour. In all three model organisms, the CNSappears to play an endocrine role in controlling or co-ordinatingthe release of insulin-like peptide, which so far have in generalbeen implicated as positive regulators of IIS, and therefore, ifanything, act to shorten lifespan [5,10]. On the other hand, theoften neuroprotective effect of IIS in the CNS itself [38,39]presents an apparent paradox, since lowered IIS activity can bothcompromise the integrity of the CNS and cause lifespan extension.Thus the impact of lowered IIS on brain aging and function isreceiving increasing attention. In the present review, we discusswhat is known about the role of the CNS in IIS-related lifespanextension in model organisms. We consider its endocrine role,and the way in which cell-autonomous effects in the CNS itselfaffect both neuronal and whole-organism survival and health. Wealso discuss the probable future directions of work in this area,including the implications of the findings for improving healthduring aging in humans.

IIS AND AGING IN MODEL ORGANISMS

Before turning to the CNS, we will provide a brief summaryof IIS and its discovery as an evolutionarily conserved pathwaydetermining lifespan. The intracellular pathway (Figure 1), as sofar identified, is considerably more complex in mammals than inthe worm and the fly. In the two invertebrates, it consists of asingle insulin-like receptor, multiple insulin-like peptide ligandsand mainly single isoforms of the other signalling components.In contrast, mammals have one insulin and two IGF ligands,which regulate the activity of the insulin and IGF-1Rs (IGF-1receptors) respectively. The IGF-1R and the IR, which has twoisoforms, are able to form homo- and hetero-dimers, resulting insix types of receptor, although the physiological significance ofeach is still unclear [40]. Along with multiple isoforms of thedownstream signalling components, which can also have tissue-specific expression patterns, there is potential for many layersof IIS complexity in mammals [41]. In all three organisms, theIIS pathway intersects with other signalling pathways, often withmore than one type of regulatory interaction and at more than one

point of the pathway. Two of these interacting pathways, TOR (tar-get of rapamycin) and JNK (c-Jun N-terminal kinase), which havebeen implicated particularly in response to nutrition and stressrespectively, are illustrated in Figure 2. The highly evolutionarilyconserved TOR pathway regulates protein synthesis and growthin response to amino acids and growth factors [42]. Mutations thatlower the activity of this pathway can extend lifespan in wormsand flies [43,44], and IIS interacts at several points with TOR sig-nalling to control lifespan in these organisms [43–45]. Increasedsignalling in the JNK pathway, which is activated by stressesincluding UV radiation and oxidative stress [46], can extend life-span [47,48]. JNK phosphorylates many transcription factors in-cluding the key IIS effector, the forkhead transcription factorFOXO, thus antagonizing IIS by causing nuclear localization ofFOXO.

The IIS pathway was first discovered to be involved in thedetermination of lifespan in C. elegans. A screen for enhancedlongevity identified the age-1 mutation which was later mappedto the catalytic subunit of PI3K [11,12,49]. Mutation of theworm IR, encoded by the daf-2 gene, also extended lifespan,and this extension required the presence of the worm forkheadtranscription factor DAF-16 [6,50–52]. In Drosophila, theinvolvement of IIS in lifespan was discovered when mutationsin the single fly IR (termed dINR) and IRS, chico, were shownto extend lifespan [13,15]. The evolutionary conservation inmammals of the control of lifespan by the IIS pathway wasconfirmed when it was found that disruption of either the insulinor IGF pathways could extend the lifespan of mice [14,17,35,36].This discovery has opened up the use of the much simpler andshorter-lived invertebrate organisms to understand the role of IISin mammalian aging. Several types of cellular defence mechanismhave been implicated in mediating the effects of IIS on lifespan,including xenobiotic detoxification [53–56], reduced proteinsynthesis [57–59], autophagy [60], proteasomal activity [24,61]and enhanced immunity [17,18,21,62,63]. The perturbationsof the IIS pathway that can extend lifespan, the downstreamsignalling effectors involved, such as the forkhead transcriptionfactors, and the candidate downstream biochemical mechanismshave been extensively reviewed recently [62,64–66].

THE CNS AND THE DETERMINATION OF LIFESPANBY LOWERED IIS

Individual tissues can contribute to the extension of lifespan byproducing secreted factors, such as the insulin ligands, that can actat a distance (endocrine) or locally (paracrine), or on the secretingcells themselves (autocrine). In addition, tissue-autonomous res-ponses to lowered systemic IIS can occur. These may either resultin improved functioning of the tissue itself, which could contributeto the survival of the whole organism (a cell-autonomous mech-anism), or could cause the tissue to emit secreted factors. Theevidence so far suggests that the role of the CNS in IIS-relatedlifespan extension is predominantly endocrine, but that cell-autonomous effects may also play a role.

Endocrine regulation of lifespan

Worm, fly and mouse CNSs differ in structure, organization andcomplexity, but, in all three model organisms, neuronal tissuereleases secreted factors that directly or indirectly modulate IISin distant tissues. Simplified models for this endocrine control oflifespan are outlined in Figure 3, which illustrates how the CNSand other endocrine tissues interact to regulate IIS at a distance andthus co-ordinate aging in the whole organism. The evidencesupporting these models in each organism, the upstream control


Insulin/IGF-like signalling, the central nervous system and aging 3

Figure 1 The IIS pathway in worms, flies and mice

Signalling begins with the binding of an INS to an appropriate receptor which induces receptor dimerization and autophosphorylation of the cytoplasmic domain. Mice and other mammals havethree ligands (insulin, IGF-1 and IGF-2), whereas worms have 38 ligands and flies have seven ligands identified so far. In contrast, worms and flies have one receptor, whereas mice have three whichadditionally can form heterodimers. The signal is then transduced either directly from the receptor (in worms and flies) or indirectly via the IRS (in worms, flies and mice) to PI3K (or AGE-1 in worms).Worms and flies have a single IRS, whereas mice encode four (IRS-1–IRS-4). PI3K converts PIP2 [phosphatidylinositol (4,5)-biphosphate] into the second messenger PIP3 [phosphatidylinositol(1,4,5)-triphosphate], and this activity is antagonized by the PTEN phosphatase (DAF-18 in worms). Elevated levels of PIP3 result in activation of PKB and PDK, and PDK then further phosphorylatesPKB, fully activating it. Flies have a single PKB, whereas worms have Akt 1, Akt 2 and SGK-1 (serum- and glucocorticoid-induced protein kinase-1), and mice have Akt 1, Akt 2 and Akt 3. Acti-vated PKB phosphorylates the forkhead transcription factor (Forkhead TF) resulting in its exclusion from the nucleus, and thus its inactivation. Worms and flies contain a single forkhead transcriptionfactor (DAF-16 and FOXO respectively), whereas mice contain three [FKHR (Forkhead homologue 1), FKHRL1 (FKHR-like 1) and AFX (FOXO 4)]. Species-specific names for each pathway componentand whether they are singly or multiply encoded are indicated in the Figure.

of ligand release and the interactions with other endocrine tissuesand hormones are described.

Mammals

In mammals, the evidence supporting the endocrine regulation oflifespan by IIS is abundant, although largely indirect. The hypo-thalamus in the mammalian brain is central to the regulationof the somatotropic axis [GH (growth hormone)–IGF-1–insulin)and the integration of many nutritional signals. The insulin-likeligands, insulin and IGF-1, are mainly produced by the pancreasand liver, with only a minor contribution from the brain [67].Repression of the somatotropic axis controlling their release

extends lifespan in several mouse models and other mammalianspecies (for a review, see [65]). For example, Snell and Amesdwarf mice both have mutations that result in lack of GH andother pituitary hormones resulting in similar phenotypes such assmall size and female sterility, and both are consistently long-lived[68,69]. Mice with defects in GH function are also long-lived. Both Lit/Lit mice, which have a mutation in the GHRHR(GH-releasing hormone receptor), but no apparent effects on otherpituitary hormones, and mice with a complete knockout of the GHreceptor/binding protein gene, are long-lived [63,70]. Althoughthese animals differ in some phenotypes due to their different hor-monal profiles, they share several traits including reduced IGF-1signalling, enhanced peripheral insulin sensitivity and glucose



Figure 2 Pathways interacting with IIS

(1) An evolutionarily conserved nutrient-sensing cascade involving the TOR kinase interacts withIIS at multiple points in the pathway. Nutrients, predominantly amino acids, regulate the activityof TOR which activates the S6 kinase (S6K), which is involved in the control of translation, celland organismal growth and cancer. Activated PKB activates the TOR pathway via its inhibition ofTSC1–TSC2 (tuberous sclerosis complex 1 and tuberous sclerosis complex 2). TOR signallingvia S6K also negatively feeds back on to IIS via an inhibition of PI3K activation. (2) Theconserved JNK pathway is activated in response to environmental factors such as UV radiationand oxidative stress. JNK promotes nuclear localization of FOXO and activation of downstreamtarget genes, thus antagonizing IIS. Although the mechanism remains to be fully elucidated,JNK may antagonize IIS via an inhibition of IRS and/or the direct phosphorylation of FOXO [48].INR, insulin receptor.

tolerance which are thought to be important for lifespan extension[64]. Mice heterozygous for an IGF-1R knockout are long-lived[14], supporting the specific role for IGF-1 in the determination oflifespan in mammals. The hypothalamus has thus been suggestedto be the site of the endocrine regulation of lifespan in mammals[71].

Fruit flies

Drosophila is an important model in the study of the endocrineregulation of aging in part because it has endocrine tissues thatresemble those of mammals (for a review, see [72]). mNSCsin the pars intercerebralis region of the fly brain produce threeof the seven DILPs (Drosophila insulin-like peptides) and havebeen suggested to be functionally equivalent to pancreatic β-cellsas producers of circulating insulin-like peptides for metabolichomoeostasis [4]. In fact, gene-expression patterns and the celllineages giving rise to the mNSCs are very similar to those givingrise to the β-cells of mammals, and there are similarities in devel-opment between the mNSCs and the mammalian anterior pituitaryand hypothalamic axis [73].

Genetic ablation of the mNSCs results in lifespan extensionpresumably by reducing levels of circulating DILP-2, -3 and -5[10]. Other studies in the fly have supported this endocrinerole of the mNSCs as the source of insulin-like peptides thatregulate aging in peripheral tissues. Expression of a dominant-negative form of p53 in the CNS using a pan-neural elavGALdriver was sufficient for lifespan extension [74], but it wasnot the case that this was due to an inhibition of neuronalapoptosis as might be expected given the role of p53 in thisprocess [75]. Instead, expression in the DILP-producing mNSCsalone was sufficient to extend lifespan, which correlated withlowered levels of dilp2 transcript in the mNSCs and loweredPI3K activity in the periphery [76]. Similarly, increases in the

stress-sensing JNK signalling pathway in neurons using the samepan-neural driver was found to be sufficient to extend lifespan andreduce the accumulation of oxidative damage in neurons [47].Again, however, expression specifically in the DILP-producingmNSCs was sufficient for the lifespan extension probably via theregulation of dilp expression [48].

Nematode worms

In the worm, multiple insulin-like peptides (INS) are expressed inmany tissues throughout the body and act as receptor agonists orantagonists [77,78]. Although this widespread expression couldsuggest that INS have paracrine or autocrine effects in the tissuein which they are expressed, some INS-expressing tissues play anendocrine role in IIS-related lifespan extension.

Specific sensory neurons have been found to influence lifespanin a DAF-16/FOXO-dependent manner [79]. Ablation of a subsetof gustatory neurons resulted in lifespan extension that was foundto be dependent on daf-16 and had no effect in a daf-2 (encodingthe worm IR) mutant, suggesting that these neurons affect lifespanby modulating IIS in distant tissues. In contrast, the lifespan ex-tension due to ablation of olfactory neurons was found to beonly partially dependent on daf-16, suggesting the involvementof unknown secreted factors. Although INS production has notbeen directly examined, these sensory neurons are thought to bethe source of INS to regulate IIS in distant tissues [79].

Two tissues, the CNS and intestine, both respond to IIS andcontribute a further level of endocrine signalling that involves thesecretion of diffusible factors, possibly INS and unknown factors,to regulate DAF-16 throughout the animal. Mosaic worms whichwere composed of wild-type and mutant daf-2 cells were long-lived despite containing many wild-type daf-2 cells, suggestingthat secondary factors were released that in turn controlled life-span [80]. Most of these mosaics contained at least some neuronaldaf-2-mutant cells, and a specific role for neurons was later con-firmed in a study which found that expression of wild-type age-1(PI3K) or daf-2 in neurons, but not in intestine or muscle, couldcompletely rescue the extended lifespan phenotype of age-1-or daf-2-mutant animals [33]. That other tissues in the body couldnot respond with altered IIS because they were mutant, and thatsuch overexpression in a wild-type animal did not shorten lifespan,supported the notion that the lifespan extension of age-1 or daf-2mutants was due to lowered IIS in neurons alone and that diffusiblefactors other than insulin-like ligands were secreted by neuronsto control lifespan [33].

In contrast, a study analysing the requirement for DAF-16activity in specific tissues using various genetic methods reportedthat DAF-16 activity in neurons accounts for only a small part ofthe lifespan extension of daf-2 mutants, and a far greater effect wasdue to expression of DAF-16 in intestinal cells [34]. This studyfurther suggested that DAF-16 activity in the intestine, and to alesser extent in neurons, controlled the production of a secondarysignal that regulated DAF-16 activity in distant cells. The discre-pancy in how much of a contribution neuronal cells made to thecontrol of lifespan in these two studies could have been due tonon-specific effects of the various trangenes used, the modulationof different IIS components (i.e. age-1, daf-2 or daf-16), orvariable endocrine effects of the tissue-specific manipulationsdepending on the genotype of the rest of the worm. More recently,a further analysis of the tissue-restricted and endocrine effectsof IIS using tissue-specific expression of new, wild-type age-1transgenes to rescue the extended lifespan of age-1 mutant wormsand a wild-type daf-16 transgene to rescue the shortened life-span of daf-16/age-1-mutant worms was performed [37]. Expres-sion of the wild-type age-1 transgenes either in different subsets



Figure 3 Models for the endocrine regulation of aging by IIS in worms, flies and mice

(A) In worms, an endocrine pathway resulting from IIS in a number of different tissues is involved in the regulation of lifespan. Sensory neurons (gustatory and olfactory) express ins genes andare thought to secrete INS and other factors in response to food and unknown environmental cues to regulate lifespan in both a DAF-16-dependent and non-dependent fashion in peripheral tissues[79]. Two tissues, the CNS and intestine, subsequently respond to IIS and produce putative secondary signals, which could include other INS and unknown non-INS factors, to regulate DAF-16activity throughout the body. The gonad is also involved in the determination of lifespan via the regulation of DAF-16 activity in the intestine. (B) DILPs 2, 3 and 5 produced by mNSCs in the parsintercerebralis region of the brain are thought to be released into the circulatory system via neuronal contacts at the Drosophila heart [3] and activate the IR in peripheral tissues. Expression of theseDILPs is regulated by nutrient intake, JNK, p53 and sNPF. Fat tissue [both the pericerebral and abdominal fat bodies (FB)] is a direct target of the DILPs; down-regulation of IIS via expression ofdFOXO in fat extends lifespan [30,31] suggesting that both fat tissues produce secondary humoral factors that regulate aging in peripheral tissues. Pericerebral fat body IIS in turn can feedback onto the mNSCs via an unknown secreted factor to down-regulate dilp expression in the mNSCs. Two other tissues have been implicated in the endocrine regulation of aging. The adult ring glandcomprises the corpora cardiaca which produces AKH (adipokinetic hormone) that is analogous to mammalian glucagon, and the corpora allata which produces JH, reduced levels of which sometimescorrelate with lifespan extension in IIS mutants, although direct evidence for its role is lacking. The mNSCs make direct contacts with the ring gland providing a possible neural control of JHexpression by the mNSCs. The female ovary produces the steroid hormone ecdysone (20E), reduced levels of which have been implicated in the control of lifespan [155]. Although a direct controlby IIS of 20E has been demonstrated in larvae [156], a link between IIS and 20E in adults has not yet been shown. (C) A very simplified model is provided to indicate that the CNS is central to thecontrol of the release of GH and its downstream effects including regulation of IGF-1 levels. Food intake results in the hypothalamus promoting hormone release from the pituitary. The hypothalamusalso produces NPY which feeds back to regulate food intake. The pituitary hormones, including LH (luteinizing hormone), FSH (follicle-stimulating hormone) and GH, cause increases in steroid sexhormones from the gonad and IGF-1 predominantly from the liver. Food intake also results in increased insulin output from the pancreatic β-cells. The circulating insulin and IGF-1 then activate IISin peripheral tissues. Adipose tissue may be important in lifespan extension due to reduced IIS, although the mechanism is not known. Mice with a fat-specific knockout of the IR (FIRKO mice) arelong-lived, have reduced fat mass and do not show age-related obesity and metabolic changes although their food intake is normal [157]. In each model, broken lines indicate speculative elementsand red stars indicate IR/IGF-1R-mediated signalling.

of neurons or in the intestine could rescue the lifespan of age-1-mutant animals in a graded fashion, suggesting that cells in thesetissues could act equally to produce endocrine factors. DAF-16had less effect on the lifespan of daf-16/age-1-mutant animalswhen expressed in either the CNS or the intestine than whenexpressed in both tissues together, suggesting that the responseof multiple tissues to IIS is required for lifespan extension.

Taken together, these studies suggest a model whereby sensoryneurons release INS and other factors in response to unknownenvironmental cues to regulate IIS in distant tissues throughoutthe worm. The CNS and intestine both respond to these secretedfactors in a DAF-2/DAF-16-dependent manner to regulate thesecretion of secondary diffusible factors that in turn regulateDAF-16 independently of DAF-2 in distant tissues. Although theidentities of the diffusible factors are unknown, JNK and HSF(heat-shock factor)-1 can regulate DAF-16 in a DAF-2-indepen-dent manner, and it has been suggested that interactions betweenproteins regulating these pathways and IIS allow for the complexco-ordination of DAF-16 activity throughout the worm [37].

Upstream factors regulating insulin-like ligand secretion

To what do the insulin-like peptide-producing cells respond toregulate the secretion of insulin-like ligands in the invertebrate

model organisms and how does this compare with the control ofthe mammalian somatotropic axis?

As described above, a role for olfactory and gustatory neuronsin the endocrine control of lifespan in worms has been identified,but the environmental factors regulating them and the identity,roles and target tissues of the INS released by these neuronsare unknown. In the fly, a role for gustatory sensory neurons inIIS-related control of lifespan has not yet been tested. However,signalling in olfactory neurons has been found to modulatelifespan, indicating evolutionary conservation of this olfactorymechanism [81]. Exposure to yeast odorants reduced the lifespanof dietary-restricted flies and mutation of an odorant-receptormolecule severely disrupted olfaction and extended lifespan infully fed flies [81]. Although the dependence on FOXO was nottested, no changes in dilp or a FOXO target-gene expression weredetected in the odorant-receptor mutant, suggesting that, similarlyto worms, olfactory control of aging in flies may be, to a largeextent, IIS-independent.

The DILP-producing mNSCs of flies, however, have been foundto be regulated by a number of factors, some of which suggestevolutionary conservation with mammals. As described above,dilp2 expression is sensitive to various stresses via p53 and JNKsignalling. In addition, the mNSCs have been shown to respond tonutrient intake to control the expression of dilp5, but not dilp2 anddilp3 [5]. Interestingly, dilp2 and dilp3, but not dilp5, expression



in the mNSCs and IIS in peripheral tissues have been found tobe regulated by sNPF (short neuropeptide F), the Drosophilaorthologue of mammalian NPY (neuropeptide Y) [82]. Similarlyto NPY, sNPF is expressed in the CNS and regulates food intake,growth, metabolism and lifespan [83]. Rat insulinoma (INS-1)β-cells similarly up-regulate insulin expression in response toNPY administration, further supporting the evolutionary con-servation of NPY/sNPF regulation of insulin-like ligand ex-pression between flies and mammals [82]. DILP2 secretion by themNSCs has been found to be controlled by serotonergic neurons tocontrol growth [84]. A connection between 5-hydroxytryptamine(serotonin) and IIS has also been observed in mammals, where5-hydroxytryptamine had effects on insulin secretion by thepancreas in rat and mouse models [85] and on IGF-1 release fromhuman granulosa cells [86]. Thus, similarly to the hypothalamusin mammals, the mNSCs appear to be the site of the integrationof many signals to control insulin-like ligand secretion andperipheral IIS.

Other endocrine tissues and secondary factors

Other hormonal pathways and endocrine tissues appear to playroles in mediating the lifespan-extending effect of reduced IIS.Spatial analysis has identified fat as an important tissue for IIS-re-lated lifespan extension. Fat-specific knockout of the IR in mice[35] and fat-specific overexpression of dPTEN or dFOXO inflies [30,31] can extend lifespan. As described above, DAF-16activity in the intestine of worms results in longevity effects andthis tissue serves as a fat storage organ [34,37]. The endocrinerole of the intestine in worms is described in detail above. In flies,both head and abdominal fat-body expression of dFOXO ex-tended lifespan [30,31]. Head fat-body expression of dFOXOresulted in lowered dilp2 expression in the mNSCs suggestingthat the increase of lifespan occurred via feedback by dFOXOto regulate dilp expression in the mNSCs [30]. In contrast, dilpexpression was not affected by abdominal fat-body expression ofdFOXO [32]. It is therefore not clear whether lifespan extensiondue to lowered IIS in fat tissue is mediated by a fat-body signal thatdown-regulates dilp expression [30] or by some other mechanism[32]. The evolutionary conservation of fat tissue in IIS-relatedlifespan extension is thus well established. In all three organisms,it is likely that secondary signals are released by fat tissue inresponse to lowered IIS, but the identity of these putative signalsand the downstream effectors and mechanisms are unknown.

The gonad has been implicated in regulating the effect ofsecreted insulin-like ligands on distant tissues. Removal of thegermline in the worm extended lifespan via an effect on DAF-16activity in the intestine [34,87], and in the fly it extendedlifespan and lowered IIS as shown by up-regulation of FOXOtarget genes [88]. Although these studies demonstrate theevolutionary conservation of the gonadal regulation of lifespan,it is probably not via the control of insulin-like peptide release atleast in flies. In germline-less flies, dilp expression was actuallyincreased, suggesting that the reduced IIS observed was due to amodulation of insulin sensitivity by the gonad [88]. Interestingly,transplantation of the ovaries of young mice into old femalesextended the remaining lifespan of the recipient [89], supportingthe evolutionary conservation of the role of the gonad inintegrating lifespan and reproduction. As reduced reproductionis not required to extend lifespan, but is often correlated with it,a goal of future studies will be to determine how IIS can affectlifespan and reproduction separately and co-ordinately.

In flies, other secondary signals such as the lipophilic hormones,ecdysone and JH (juvenile hormone) [9,90], which play importantroles during insect development, have been suggested to be

involved [72]. Ecdysone in adult flies is produced in the ovarianfollicle cells and adult-specific ecdysone signalling has beenfound to modulate lifespan in a sex-specific fashion, but it is notyet known if it and reduced IIS affect lifespan by overlapping ordistinct mechanisms [90]. JHs are major developmental regulatorsin insects which are also involved in adult female vitellogenesis,oogenesis and entry into reproductive diapause in response tochanges in photoperiod or temperature. Although some studiessuggest that JH levels are involved in the regulation of lifespan[91,92], low JH production does not always correlate with life-span extension in IIS mutants [93]. The Drosophila IR isexpressed in the JH-producing gland (corpora cardiaca/corporaallata) of adult Drosophila and the mNSCs make axonal contactswith this gland, such that IIS has the potential to regulate JHproduction, but further experiments are needed to confirm a directrole in lifespan determination.

Summary

Recent advances in aging research have confirmed the neuro-endocrine regulation of IIS-related lifespan extension but manyquestions remain, including the following. (i) Which specificinsulin-like peptides modulate IIS in each responsive tissue?(ii) What are the cues and mechanisms controlling their secretionand action? (iii) What are the roles of binding proteins in targetingand/or modulating the activity of the insulin-like peptides?(iv) What are the secondary factors released in response to IISby other endocrine tissues?

Next, we consider the CNS as a tissue that responds to IIS anddiscuss how cell-autonomous effects in the CNS itself affect bothneuronal and whole-organism survival and health.

Cell-autonomous effects of IIS in the CNS

Although the brain has long been thought to be insulin-insensitive,the IIS pathway has been found to play roles in many aspects ofCNS development and function in diverse organisms, includingaxonal guidance [94], neurogenesis [38], neuronal survival andprotection from apoptosis [39] and cognition [95]. Thus the CNSis also a downstream target of IIS with cell-autonomous responsesthat could affect neuronal survival and neurodegenerative disease,and CNS function. These processes could all ultimately affect theaging of the whole organism.

The role of IIS in neuronal survival and CNS function

In mammals, adult brain function and integrity require a constantinput of trophic factors from local (e.g. glial cells) and peripheralsources including steroid hormones and growth factors suchas insulin and IGF-1 that cross the blood–brain barrier [96].Both insulin-like ligands are neurotrophic since they can supportneuronal growth, survival and differentiation in the absence ofother growth factors [67]. Insulin and IGF-1Rs are expressedthroughout the brain, but most abundantly in specific regions suchas the cerebral cortex, hippocampus, thalamus, hypothalamus,spinal cord and retina [97]. In the adult CNS, the activation of IISby insulin and IGF-1 is stimulatory to neuronal survival via theaction of PI3K to directly inhibit apoptosis [98,99]. The mech-anism involves the activation of PKB (protein kinase B; alsoknown as Akt), which protects against apoptosis by inactivatingproteins involved in the apoptotic machinery [100,101], inhibit-ing GSK-3β (glycogen synthase kinase-3β) [102], inactivatingdownstream transcription factors, such as FOXO [103], and down-regulating the transcriptional activity of p53 [104]. In addition



to promoting the survival of neurons, IIS is beneficial to CNSfunction. The IR is involved in synaptic plasticity [105,106]and plays a modulatory role in learning and memory [107].Signalling via the IR is also involved in the maintenanceof synapses that contributes to brain circuit formation andexperience-dependence plasticity [108], and mice with reducedIGF-1 levels showed impaired performance of a hippocampal-dependent spatial memory task [95].

However, the roles and mechanisms of action of insulin andIGF-1 in the CNS have not been fully elucidated and it appearsthat both ligands can in some circumstances be inhibitory toneuronal survival. High levels of insulin in the brain are associatedwith neurotoxicity, which is thought to be due to increased pro-duction of free radicals and elevated oxidative stress [109]. Insulinhas also been found to induce neuronal cell death at physiologi-cally high or low glucose concentrations in an in vitro rat hippo-campal culture system [110]. Likewise, IGF-1 has been proposedto be capable of inhibiting neuronal survival in some circum-stances, via the negative regulation of SIRT (sirtuin) 1 expression[111,112]. Sirtuins are NAD+-dependent protein deacetylases thatregulate gene expression (for reviews, see [113–115]), and theiroverexpression in yeast and worms has been found to extendlifespan, which in the worm requires the FOXO transcriptionfactor [116,117]. In mammals, of the seven members of the Sir2family, SIRT1 is the closest to yeast Sir2 [118] and has beenshown to have a protective effect on the survival of neuronalcells [119]. The balance of positive and negative effects of IGF-1 on neuronal survival would thus depend on metabolic statusand NAD levels [111]; however, the mechanism by which IGF-1regulates SIRT1 and its relevance to neuronal survival is unknown.SIRT1 itself has also recently been shown to have both pro- andanti-aging functions [120]. Inhibition of SIRT1 in mice resultedin a shortened lifespan, but also decreased IGF-1 signalling andenhanced resistance to oxidative stress of neuronal cells [120].Sirtuin biology adds another layer of complexity that will affectthe outcome of IIS manipulations on neuronal survival.

Impact of lowered IIS on natural neurodegeneration and functionalsenescence

The study of natural age-related neurodegeneration and its re-lationship to age-related functional decline (functional senes-cence) and IIS is in its infancy. In humans, normal aging isassociated with a decline in plasma levels of IGF-1 [121], whichis associated with neuronal aging and neurodegeneration, andeven healthy older people tend to show mild cognitive deficits[96]. In contrast, plasma insulin levels tend to increase with agedue to progressive peripheral insulin resistance which can result inneurotoxic levels of insulin in the brain [109,122]. Thus reducedIIS in the CNS resulting from lower levels of peripheral IGF-1and increased IIS due to hyperinsulinaemia during normal agingare both associated with brain senescence in humans [123].

Results are beginning to emerge from studies in the modelorganisms that specific IIS genetic manipulations can amelioratesome forms of behavioural senescence, suggesting that they havethe potential to reduce neurodegeneration or improve neuronalfunction with age. For instance, although the nervous system ofC. elegans shows few signs of degeneration during normal aging,old animals do display behavioural defects [124] that could in partbe due to compromised CNS functioning at late ages. Long-livedage-1 and daf-2 mutants show improved thermotaxis learningbehaviour, a type of associative conditioning, with age which wassuggested to be due to increased resistance to neuronal stressesand disease [20,125]. Changes in locomotor activity did not appearto be involved, suggesting that the improved learning behaviour

of these animals was due to enhanced associative conditioning.However, the long-lived animals did show increased thermalperception compared with controls, which could have contributedto their improved thermotaxis-learning behaviour.

In Drosophila, although gross changes in CNS integrity withnormal aging have also not been reported, specific neuronalcell types have been found to degenerate with age, which cor-relates with the senescence of experience-dependent behavioursmodulated by these neurons [126], and flies show AMI (age-related memory impairment) [127]. The chico1 mutation in flieshas been shown to slow age-related decline in motor function[19], suggesting that lowered IIS has the potential to ameliorateCNS-based declines. However, the functional measure used inthe study was negative geotaxis and it is not known whether theimprovement in this behaviour was due to attenuated age-relateddegeneration of the CNS or in other organ systems required forclimbing ability, such as muscle.

Some mammals with reduced activity of the somatotropicaxis, such as long-lived Ames dwarf mouse and the GHRKO(GH receptor knockout) mouse [68,128], have improved memoryretention with age [129,130]. For the Ames mouse, these improve-ments in cognitive function could be due to increased localproduction of IGF-1 that has been found to occur in the hippo-campus in response to the low circulating levels of this ligand[131]. Interestingly, long-lived mNSC-ablated flies show anattenuated decline in negative geotaxis behaviour and increasedlevels of dilp4 (S. Broughton and L. Partridge, unpublished work).This dilp is not expressed in the mNSCs, and preliminary datasuggest that it is expressed at low levels in the brain. Thus someIIS manipulations may result in specific compensatory increasesin insulin-like ligands that protect the CNS from the neurotoxiceffects of reduced systemic IIS.

Given the neurotoxic effect of age-related hyperinsulinaemiain mammals, it is also possible that reducing the expression oractivity of specific components of the IIS pathway that mediatethe response to insulin in the mammalian brain could have neuro-protective effects that ameliorate natural neurodegeneration. Thishypothesis is supported by the finding that a brain-specificknockout of IRS-2 in mice was sufficient for lifespan extension[36]. However, it is not known whether these mice are in factprotected from age-related neurodegeneration or whether theydisplay any improvements in brain function with age comparedwith control animals.

Impact of lowered IIS on neurodegenerative disease

The dysregulation of IIS in the CNS is also thought to play a majorrole in the pathology of neurodegenerative diseases, which areassociated either with low serum IGF-1 levels or with high levels,resulting in IGF-1 resistance. For example, most results indicatethat insulin and IGF-1 resistance are linked to the development oflate-onset forms of Alzheimer’s disease and neurodegenerationassociated with Type 2 diabetes (for reviews, see [132–135]).Many potential mechanisms may exist to mediate this connection,which is in general poorly understood, but several studies havesuggested that insulin and IGF-1 may directly affect the molecularpathologies underlying neurodegenerative disease. On the onehand, some studies suggest that insulin and IGF-1 signallingmay protect against such pathologies. Insulin and IGF-1 are bothinvolved in the control of β-amyloid metabolism, involved inthe pathogenesis of Alzheimer’s disease in a complex manner,and it has been suggested that the pathogenic event triggeringAlzheimer’s disease is the development of IGF-1 resistance at theblood–brain barrier [132]. Furthermore, the hippocampus ofthe long-lived Ames mice, which has been shown to produce



IGF-1 locally, was found to be resistant to the deleterious effects ofβ-amyloid peptide [136]. On the other hand, more recent studiessuggest that reductions in insulin signalling which extend lifespanmay actually confer a protective effect against abnormal proteinaggregation in neurodegenerative disease. In a worm model ofAlzheimer’s disease, lowered IIS resulted in reduced toxicityof the Aβ-(1–42) [amyloid β-peptide (1–42)] via the regulation ofthe downstream transcription factors HSF-1 and DAF-16, whichmediate cellular detoxification mechanisms [24]. Modulation ofthe IIS pathway could also affect neurodegenerative disease by itsregulation of TOR kinase, which has been shown to modulate theneurotoxicity of aggregrate-prone proteins such as polyglutamineexpansions and mutant tau that are involved in fly and mouseneurodegenerative disease models [137,138]. Hence the effects ofinsulin and IGF-1 dysregulation on neurodegenerative processesappear to be complex and may have positive or deleteriouseffects, possibly depending upon the level and mechanism ofdysregulation.

Impact of lowered IIS in the CNS on whole-organism aging

The cell-autonomous effects of lowered IIS in the CNS, describedabove, whether beneficial or deleterious to neuronal survival,could affect the survival of the whole organism via an effecton the integrity and functioning of the CNS with age. Before wediscuss the evidence that beneficial effects on neuronal survivalin the CNS contribute to IIS-related lifespan extension, it shouldbe noted that the relationship between neurodegeneration andlifespan is far from clear. For instance, although neurodegener-ation is usually associated with shortened lifespan [139], aDrosophila mutant displaying neurodegeneration and behaviouraldeficits was found to have a normal lifespan [140]. This suggeststhat the two processes can be uncoupled in some circumstancesand raises the possibility that some degree of neurodegenerationor reduced CNS function could occur in long-lived IIS mutantswithout impinging on the lifespan of the organism.

Some long-lived IIS mutants, however, have been found to showimproved cognitive or behavioural function with age, such asthe Ames dwarf mouse described above, suggesting that loweredperipheral IIS can have beneficial effects on the CNS; however,it is not known whether these putative effects in the CNS arerequired for or contribute to the extended lifespan of these animals.In worms, lowered IIS in neurons alone was suggested to be suffi-cient for lifespan extension in one study [42], and in another itwas found to have an effect in combination with lowered IIS inother tissues [37]. Despite some discrepancies, these studies sug-gested that endocrine outputs from the CNS played a large partin the lifespan extension, and it is not known to what extent cell-autonomous responses to lowered IIS in neurons contribute. Inflies, there is as yet no direct evidence to suggest that down-regulating IIS in the CNS has cell-autonomous effects that areeither sufficient for or contribute to lifespan extension.

The most direct evidence comes from the Irs2 brain-specificknockout mice. The global knockout of Irs2 resulted in shortenedlifespan due to progressive diabetes [17,141], but when the de-letion was brain-specific, the mice were long-lived despite theobesity and insulin resistance that also resulted from this mutation[36]. As described above, these Irs2 brain-specific knockout micewere suggested to be long-lived because they were protectedfrom the neurotoxic effects of age-related increases in insulinlevels [36]. If true, this would suggest that enhanced neuronalsurvival or function due to lowered IIS can contribute to lifespanextension in some circumstances. However, the peripheral effectson insulin resistance and obesity of the Irs2 brain-specificknockout [36] raises the possibility that altered neuroendocrine

function could play a part in the lifespan extension. Indeed, IRS-2 in a subpopulation of hypothalamic neurons plays an insulin-responsive role in energy homoeostasis and growth [142], and theactivity of the IR in the brain has been shown to regulate whiteadipose tissue mass and glucose levels in mice [143].

Interestingly, in contrast with the global knockout of IRS-2 [36],homozygous global deletion of Irs1, which is also expressed in thebrain, extended the lifespan of mice and improved some aspectsof health, such as glucose homoeostasis and markers of skin,bone, immune and motor function [17]. It will be very interestingand important to determine the cognitive status of these mice aswell as the brain-specific Irs2-knockout mice [36] as they ageunder variable environmental conditions. Taken together, theseresults reflect the tissue-specific roles and differential couplingto downstream signalling components of the IRS proteins inmammals [41], and indicate that modulation of specific IIS com-ponents can either contribute to, or be sufficient for, lifespanextension under certain conditions. However, the degree towhich enhanced neuronal survival or function due to altered IIScontributes to lifespan extension is still unclear.

Putative CNS-specific mechanisms contributing to IIS-related lifespanextension

If we assume that in some circumstances lowered IIS cancontribute to lifespan extension by promoting neuronal survivaland functioning of the nervous system with age, what are thecandidate cell-autonomous mechanisms mediating the response?Indirect evidence has implicated autophagy. Autophagy is crucialfor cell survival under extreme conditions, by providing energyvia the catabolysis of cytoplasmic components [144]. It was firstidentified as a potential longevity assurance mechanism down-stream of IIS because it is required for the lifespan extensionof IIS-mutant worms [60], although it is not known whetherautophagy specifically in the CNS is required for the lifespanextension of these animals. In flies, reduced autophagy is asso-ciated with short lifespan and neurodegeneration [139], suggest-ing that it is required for the basic maintenance and survival ofneurons. Promoting levels of autophagy in neurons alone wassufficient to extend lifespan and increase resistance to oxidativestress, possibly via the prevention of age-dependent accumulationof damage in neurons [145]. As autophagy is predominantlyregulated by the TOR pathway, lowered IIS can induce it via thereduced action of PDK (phosphoinositide-dependent kinase) 1/PKB on TOR kinase [146]. However, the unexpected finding thata mouse model of Hutchinson–Gilford progeria showed extensivebasal activation of autophagy and metabolic changes usuallyassociated with lifespan extension suggests that chronic activationof this pathway can be pro-aging [147], and that an optimal,intermediate level of autophagy is required for lifespan extension.

Comparative microarray analysis of long-lived IIS mutantanimals has identified a number of other evolutionarily conservedmolecular processes, including xenobiotic detoxification, reducedprotein synthesis and enhanced immunity, which are candidatesfor mechanisms mediating IIS-related lifespan extension [62].Little direct experimentation has so far been performed to testthe array-based predictions that these mechanisms are sufficientto extend lifespan, but it was recently shown that up-regulationof Keap1 (Kelch-like ECH-associated protein 1)/Nrf2 (nuclearfactor-erythroid 2 p45 subunit-related factor 2) signalling in the flyand SKN-1 (SKiNhead 1) in the worm extends lifespan [148,149].Nrf2 transcription factor and its specific repressor Keap1 mediatescellular responses to oxidative stresses and xenobiotics in flies.Activation of signalling by reduced expression of keap1 resultedin increased tolerance to oxidative stress and extension of lifespan



[149]. In the worm, phase-two detoxification mechanisms whichprovide defence against oxidative stress are regulated by the Nrf2-related SKN-1, the expression of which is required for lifespanextension due to dietary restriction in worms [150]. SKN-1 isdirectly inhibited by IIS, mutations in SKN-1 suppressed IIS-related lifespan extensions, and increased expression of SKN-1-extended lifespan [148].

Although less is known about whether modulation of anyof these mechanisms in the CNS plays a part in promotingneuronal or whole-organism survival, some indirect evidence doesexist. Although it is not known how reducing protein synthesismight contribute to lifespan extension, it has been shown thatit confers resistance to heat stress on worms [58], leading tothe suggestion that the mechanism is connected to the activity ofprotein chaperones [62]. Ubiquitous overexpression of such HSFscan extend lifespan in both worms [151,152] and flies [153], andrecently the expression of HSFs restricted to the CNS in flieswas found to be sufficient to extend lifespan [154]. Alternatively,reduced protein synthesis may result in an increase in autophagy,up-regulation of which in neurons, as described above, has beenshown to be sufficient for lifespan extension [145].

CONCLUSIONS AND FUTURE PERSPECTIVES

The often beneficial effect of IIS on neuronal survival and CNSfunction has presented an apparent paradox, since lowered IIS ac-tivity has the potential to both compromise the integrity of the CNSand extend lifespan. However, the potential for IIS in the CNS tohave both negative and positive effects, together with the tissueand timing requirements and the sensitivity to environmentalconditions of the lifespan-extending IIS manipulations, suggeststhat no paradox exists. Moreover, the finding that a Drosophilamutant with significant neurodegeneration and behaviouraldeficits has a normal lifespan [140] illustrates that neurode-generation and mortality are only loosely coupled. This findingraises the possibility that IIS manipulations may not always bebeneficial to CNS integrity and functioning even when they extendlifespan, if positive effects of lowered IIS on peripheral organsystems outweigh any negative effects in the CNS.

Although some aspects of health have been shown to beimproved, the functional status with age of many long-livedanimals remains to be determined. Thus, in the short term, thereis a great need for investigations to determine the relationshipsbetween neurodegeneration, behavioural and cognitive functionand lifespan. In the longer term, the discovery of the cellular andbiochemical mechanisms mediating altered functioning of theCNS in long-lived animals will further the identification of poten-tial therapeutic targets of functional senescence and neurodegen-erative disease.

The discoveries made in worms, flies and mice on the extensionof lifespan by lowered IIS suggest that manipulation of theright signalling component in the right tissue in the adult canextend lifespan with few detrimental effects or even improvedhealth and function. Together with the identification of possiblyevolutionarily conserved tissue-specific longevity assurancemechanisms, this raises the exciting possibility that futureresearch will identify pharmacological targets and interventionscapable of extending lifespan and maintaining the function of thenervous system during aging.

ACKNOWLEDGEMENTS

We thank M. Piper, F. Kerr and M. Hodges for comments on the manuscript, andD. Withers for helpful suggestions prior to submission.

FUNDING

Our work is supported by the Wellcome Trust [grant number 066750/A/01/Z].

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Received 30 October 2008; accepted 24 November 2008Published on the Internet 28 January 2009, doi:10.1042/BJ20082102


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