Sci. Aging Knowl. Environ., 30 October 2002
Vol. 2002, Issue 43, p. pe17
[DOI: 10.1126/sageke.2002.43.pe17]

PERSPECTIVES

Of Worms, Flies, Dwarfs, and Things That Go Bump in the Night

William E. Sonntag, and Melinda M. Ramsey

The authors are in the Department of Physiology and Pharmacology at the Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA. E-mail: wsonntag{at}wfubmc.edu (W.E.S.)

http://sageke.sciencemag.org/cgi/content/full/sageke;2002/43/pe17

Key Words: C. elegansdaf-2daf-16 • insulin-like growth factor 1 • IGF-1 • life-span

Introduction

Over the past several years, it has become evident that disruption of the insulin/insulin-like growth factor (INS/IGF)-related daf-2 pathway in Caenorhabditis elegans can significantly extend life-span (see Johnson Review). The daf-2 gene encodes a member of the insulin receptor family, and previous studies have indicated that binding of an insulin-like substrate to this receptor initiates a cascade of events, including activation of a homolog of mammalian phosphoinositide-3-OH kinase (PI3K) encoded by age-1 (1). This kinase, in turn, activates the akt- (pdk-1) encoded protein kinase B (PKB), which blocks the function of a forkhead/winged helix transcription factor (encoded by daf-16) by preventing its translocation to the nucleus (1) (Fig. 1). Disruption of the daf-2 pathway (by, for example, mutations in the daf-2, age-1, or akt genes) in C. elegans has been shown to extend life-span substantially, in that animals carrying such mutations live from 30 to 100% longer than their wild-type counterparts (2, 3). These effects appear to be the result of the release of the AKT/PKB "brake" on DAF-16 (4), the primary target of the daf-2 pathway whose function is required for life-span extension in daf-2 and age-1 mutants (5). DAF-16 regulates gene expression that is essential to environmental stress resistance, development, and dauer formation (6). Furthermore, components of the daf-2 signaling pathway in C. elegans share significant homology with components of metabolic pathways in flies and yeast that have also been reported to have profound effects on life-span (4, 7, 8). The similarities in INS/IGF-like signaling pathways between these organisms and mammals raise the possibility that modulation of insulin and/or IGF-1 signaling in mammals might also influence longevity. The mammalian growth hormone/IGF-1 pathway is illustrated in Fig. 2.



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 1. C. elegans daf-2 pathway. Under permissive growth conditions, an insulin-like substrate binds the daf-2-encoded receptor and initiates a cascade of events, including activation of the age-1-encoded homolog of PI3K. PI3K activates the akt-encoded PKB, which phosphorylates the DAF-16 transcription factor, preventing its translocation to the nucleus. Disruption of the daf-2 pathway (by mutations of the daf-2, age-1, or akt genes) prohibits the phosphorylation of the DAF-16 transcription factor, permitting its translocation to the nucleus. DAF-16 transcription factor function is required for life-span extension in daf-2 and age-1 mutants.

 


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 2. Growth hormone/IGF-1 pathway. Growth hormone, produced in the anterior pituitary, is modulated by two hypothalamic hormones: GHRH, which stimulates the synthesis and secretion of growth hormone, and somatostatin (SS), which inhibits growth hormone release in response to GHRH. Growth hormone binds with high affinity to its receptor, which is found in tissues throughout the body, and activation of this receptor stimulates the synthesis and secretion of IGF-1 (14). Although 90% of circulating IGF-1 is synthesized and secreted by the liver, many types of cells, including some found in the brain and vasculature, are capable of IGF-1 production (15, 16). Binding of the hormone to the IGF-1 receptor causes potent mitogenic effects, including increases in DNA, RNA, and protein synthesis (17). Although heterogeneity exists in the processing of IGF-1 mRNA, the result is a single peptide that is structurally homologous to proinsulin. Blood and tissue concentrations, as well as activity of the peptide, are regulated by IGF-1-binding proteins (IGFBPs) (17). Although it was initially proposed that all of the actions of growth hormone are mediated through IGF-1, data from several studies support direct roles for growth hormone in the regulation of lipolysis and insulin sensitivity that are independent of IGF-1 action (18, 19).

 
Confounding Variables in Interpretation

There is no doubt that mutational analysis of daf-2 signaling has had a profound impact on our understanding of the genetic regulation of life-span. However, a persistent problem in these invertebrate models has been the fact that gene function is affected throughout life, including during early stages of development. Modulation of daf-2 signaling during this crucial period results in physiological alterations that could potentially affect life-span independently, confounding investigations into the mechanisms behind the increased life-span phenotype. These confounding variables include diminished reproductive capacity (9) and impaired development of several key organ systems that are necessary for normal adult function. For example, daf-2 mutations influence both life-span and reproduction. As a result, it is difficult to determine the relative contributions of delayed reproduction and INS/IGF-1 signal impairment to the increased life-span observed in these mutant worms. In fact, some investigators have provided compelling evidence to support the conclusion that alterations in reproductive capacity alone have a direct effect on life-span (10). These and related issues have impeded progress in the understanding of specific mechanisms for life-span extension in invertebrate models and have limited the development of appropriate mammalian models. If evidence were forthcoming that suppression of the INS/IGF-1 signaling pathway solely in adults had the potential to influence life-span, however, many of these confounding variables could be controlled or eliminated. For example, both insulin and IGF-1 have important developmental effects in mammals. These hormones regulate growth and maturation of diverse organ systems, from the pancreas and gastrointestinal tract to the brain, where synaptic density is affected (11). Thus, any pertubation in the growth hormone/IGF-1 or insulin pathway throughout development (such as often occurs in transgenic animals) is likely to have widespread effects that make it difficult or impossible to separate the primary effects of the mutation from the developmental effects that are present in the transgenic animal. Conditional mutants would greatly benefit these studies, but they have not yet been developed.

Suppression of daf-2 in Adults Increases Life-Span

In their most recent publication, Kenyon and colleagues [Science 298, 830-834 (2002); see Dillin et al.] used RNA interference (RNAi) to decrease mRNA concentrations and regulate the expression of daf-2 and daf-16 in C. elegans. Because RNAi is accomplished by feeding worms bacteria that express the appropriate double-stranded RNA (dsRNA), the developmental time point at which RNAi is initiated can be controlled. The primary goal of these researchers was to determine the phase in the life-span during which the effects of daf-2 suppression are most evident, and to determine whether suppression at distinct stages differentially affects processes such as reproduction and the rate of aging. As expected, treatment with daf-2 RNAi beginning at the time of hatching and continuing through adulthood suppressed daf-2 expression and increased life-span. The investigators then showed that if daf-2 expression was suppressed from hatching until the last larval stage (L4), the reproductive period was protracted, and the effects could be reversed by RNAi suppression of daf-16. Additional experiments indicated that suppression of daf-2 expression only during larval development, but not during adulthood, did not increase life-span. Conversely, if suppression of daf-2 was initiated in young adults, life-span increased but reproduction was not affected. The life-extending effects steadily declined and became insignificant when RNAi treatment began after adult day 6. Another interesting aspect of these studies is the finding that the increased life-span of daf-2 mutants could be suppressed by initiating RNAi of daf-16 function during adulthood, demonstrating the specificity of the interventions. These studies support the conclusion that the effects of daf-2 suppression on life-span and reproduction occur through separate mechanisms. Furthermore, the results suggest that the INS/IGF-1-like daf-2 pathway in C. elegans influences dauer formation and reproduction early in life but exerts its effects on life-span exclusively during adulthood. Finally, because daf-2 also functions in stress resistance, the authors studied the effects of paraquat, an oxidative damaging agent, on animals treated with daf-2 dsRNA during adulthood. These worms were resistant to paraquat, indicating that daf-2 functions in stress resistance during adulthood. The authors conclude that this additional consequence of INS/IGF-1-like signaling suppression may contribute to the observed increases in longevity.

The fact that suppression of daf-2 signaling has effects on both reproductive function and life-span is consistent with earlier studies from this and other laboratories (9, 10). However, the temporal separation of the effects of daf-2 suppression on development, reproduction, and life-span represents a novel and significant contribution to the field.

Confounding Variables in Mammalian Studies Remain

The application of these findings to mammals presents both opportunities and challenges. Two frequently used models for studying aging in mammals, the Ames and Snell dwarf mice, exhibit life-span extension as a consequence of mutations in prop-1 and pit-1, respectively (see Bartke Viewpoint). These mutations result in deficiencies in prolactin, thyroid-stimulating hormone (TSH), and growth hormone. Reduced production of these hormones throughout development causes a number of anomalies in organ development, as well as secondary endocrine deficiencies that complicate attempts to identify the specific mechanisms responsible for the life-span extension observed in these animals. Furthermore, dwarf animals such as those produced by mutation of the Pit-1 locus must be housed with normal wild-type animals in order to demonstrate increased life-span (12), a possible consequence of altered temperature homeostasis. Whether these effects result from developmental anomalies, the interactions of multiple endocrine deficiencies, or growth hormone/IGF-1 deficiency is unresolved. The simultaneous alterations in the concentrations of prolactin, TSH [and triiodothyronine (T3)], growth hormone, glucocorticoids, insulin, and glucose, along with the compensatory mechanisms induced by such changes, make the development of specific mechanistic hypotheses related to aging and the genetics of life-span a daunting challenge. Attempts to circumvent the aforementioned problems through specific mutation of the growth hormone-releasing hormone (GHRH) receptor (see Little Mouse) and growth hormone receptor (see Growth Hormone Receptor Knockout Mouse) have resulted in developmental anomalies and secondary endocrine alterations similar to those evident in the Ames and Snell models.

Future Directions and Conclusions

If daf-2 signaling in C. elegans is homologous to INS/IGF-1 pathways in mammals, the recent work by Dillin et al. and others provides compelling evidence that antagonism of either insulin or IGF-1 signaling after puberty might influence life-span. Studies that address this issue while avoiding the developmental anomalies and multiple endocrine deficiencies are not only feasible but necessary to understand the specific biological events within the growth hormone/IGF-1 pathway that regulate mammalian aging. For example, transgenic models that exhibit specific deficiencies in the expression of IGF-1 in the liver (LID mice) (13) are available, and studies of these animals as well as transgenic animals with appropriate alterations in the IGF-1 and insulin pathways should be pursued. Each model requires the design of rigorous experiments that control for (or at least recognize) developmental anomalies and compensatory endocrine mechanisms that potentially confound the interpretation of results. Animals with conditional disruption of specific genes within the insulin and IGF-1 pathways would be of most benefit, but it will be several years before such models are fully developed. Only with well-controlled and specific studies will researchers be able to determine whether the findings in invertebrate models are applicable to mammals.

Despite much rhetoric on this topic, to date no model has demonstrated that a specific deficiency in growth hormone, IGF-1, or insulin has the capacity to regulate life-span in mammals. In many cases, the compensatory mechanisms of the endocrine system that are prevalent in mammals have been overlooked and the consequences of such changes ignored. A clear foundation has been established in invertebrate models indicating that INS/IGF-like signaling regulates aging in several species. It is now the responsibility of molecular biologists, endocrinologists, and physiologists who study mammalian systems to (i) determine whether specific manipulations of this pathway regulate aging in mammals and (ii) establish mechanisms of action.


October 30, 2002
  1. S. Paradis, G. Ruvkun, Caenorhabditis elegans Akt/PKB transduces insulin receptor-like signals from AGE-1 PI3 kinase to the DAF-16 transcription factor. Genes Dev. 12, 2488-2498 (1998).
  2. C. Kenyon, J. Chang, E. Gensch, A. Rudner, R. A. Tabtiang, C. elegans mutant that lives twice as long as wild type. Nature 366, 461-464 (1993).
  3. T. E. Johnson, Increased life-span of age-1 mutants in Caenorhabditis elegans and lower Gompertz rate of aging. Science 249, 908-912 (1990).
  4. C. Kenyon, A conserved regulatory system for aging. Cell 105, 165-168 (2001).
  5. K. Lin, J. B. Dorman, A. Rodan, C. Kenyon, Regulation of the Caenorhabditis elegans longevity protein DAF-16 by insulin/IGF-1 and germline signaling. Nat. Genet. 28, 139-145 (2001).
  6. J. Apfeld, C. Kenyon, Cell nonautonomy of C. elegans daf-2 function in the regulation of diapause and life span. Cell 95, 199-210 (1998).
  7. D. J. Clancy, D. Gems, L. G. Harshman, S. Oldham, H. Stocker, E. Hafen, S. J. Leevers, L. Partridge, Extension of life-span by loss of CHICO, a Drosophila insulin receptor substrate protein. Science 292, 104-106 (2001).
  8. P. Fabrizio, F. Pozza, S. D. Pletcher, C. M. Gendron, V. D. Longo, Regulation of longevity and stress resistance by Sch9 in yeast. Science 292, 288-290 (2001).
  9. H. A. Tissenbaum, G. Ruvkun, An insulin-like signaling pathway affects both longevity and reproduction in Caenorhabditis elegans. Genetics 148, 703-718 (1998).
  10. H. Hsin, C. Kenyon, Signals from the reproductive system regulate the lifespan of C. elegans. Nature 399, 362-366 (1999).
  11. C. S. Carter, M. M. Ramsey, W. E. Sonntag, A critical analysis of the role of growth hormone and IGF-1 in aging and lifespan. Trends Genet. 18, 295-301 (2002).
  12. K. Flurkey, J. Papaconstantinou, R. A. Miller, D. E. Harrison, Lifespan extension and delayed immune and collagen aging in mutant mice with defects in growth hormone production. Proc. Natl. Acad. Sci. U.S.A. 98, 6736-6741 (2001).
  13. J. L. Liu, S. Yakar, D. LeRoith, Mice deficient in liver production of insulin-like growth factor I display sexual dimorphism in growth hormone-stimulated postnatal growth. Endocrinology 141, 4436-4441 (2000).
  14. D. LeRoith, C. Bondy, S. Yakar, J. L. Liu, A. Butler, The somatomedin hypothesis. Endocr. Rev. 22, 23-74 (2001).
  15. J. Lopez-Fernandez, F. Sanchez-Franco, B. Velasco, R. M. Tolon, F. Pazos, L. Cacicedo, Growth hormone induces somatostatin and insulin-like growth factor I gene expression in the cerebral hemispheres of aging rats. Endocrinology 137, 4384-4391 (1996).
  16. H. Yamamoto, L. J. Murphy, Enzymatic conversion of IGF-I to des(1-3)IGF-I in rat serum and tissues: a further potential site of growth hormone regulation of IGF-I action. J. Endocrinol. 146, 141-148 (1995).
  17. W. S. Cohick, D. R. Clemmons, The insulin-like growth factors. Annu. Rev. Physiol. 55, 131-153 (1993).
  18. W. H. Daughaday, A personal history of the origin of the somatomedin hypothesis and recent challenges to its validity. Perspect. Biol. Med. 32, 194-211 (1989).
  19. O. G. Isaksson, A. Lindahl, A. Nilsson, J. Isgaard, Action of growth hormone: current reviews. Acta Paediatr. Scand. Suppl. 343, 12-18 (1988).
Citation: W. E. Sonntag, M. M. Ramsey, Of Worms, Flies, Dwarfs, and Things That Go Bump in the Night. Science's SAGE KE (30 October 2002), http://sageke.sciencemag.org/cgi/content/full/sageke;2002/43/pe17








Science of Aging Knowledge Environment. ISSN 1539-6150