Fax +41 61 306 12 34E-Mail karger@karger.chwww.karger.com

Debate

Gerontology 2007;53:306–321 DOI: 10.1159/000103924

Screening Candidate Longevity Therapeutics Using Gene-Expression Arrays

Stephen R. Spindler Patricia L. Mote

Department of Biochemistry, University of California, Riverside, Calif. , USA

However, because the mice die mostly of cancer, only che-mopreventives active against specific cancers can be identi-fied by such studies. The studies were also time-consuming and expensive. We discuss high-density microarray studies of the effectiveness of glucoregulatory drugs and putative cancer chemopreventatives at reproducing the hepatic gene-expression profiles of long-term and short-term CR. We describe the identification of one compound, metfor-min, which reproduces a subset of the gene-expression and physiological effects of CR. Conclusion: Taken together, our results suggest that gene-expression biomarkers may be su-perior to lifespan studies for initial screening of candidate longevity therapeutics. Copyright © 2007 S. Karger AG, Basel

Introduction

At present, there are no authenticated longevity phar-maceuticals. This situation does not indicate that such compounds do not exist or cannot be developed. Rather, it indicates that we have not found an effective means for identifying such pharmaceuticals. Assays involving lifes-pan studies in model organisms have practical and theo-retical limitations. Therefore, some gerontologists con-jecture that aging must be fully understood before effec-

Key Words Caloric restriction mimetics � Insulin/IGF-I signaling � Drug development � Drug discovery � Lifespan � Microarray

Abstract Background: We review studies showing that CR acts rap-idly, even in late adulthood, to extend health- and lifespan in mice. These rapid physiological effects are closely linked to patterns of gene expression in liver and heart. Non-hu-man primate and human studies suggest that the signal transduction pathways responsible for the lifespan and health effects of caloric restriction (CR) may also be involved in human longevity. Thus, pharmaceuticals capable of mim-icking the effects of CR (and other methods of lifespan ex-tension) may have application to human health. Objective: We show that lifespan studies are an inefficient and theo-retically problematic way of screening for longevity thera-peutics. We review studies suggesting that rapid changes in patterns of gene expression can be used to identify pharma-ceuticals capable of mimicking some positive effects of ca-loric restriction. Results: We present a traditional study of the effects of melatonin, melatonin and pregnenolone, aminoguanidine, aminoguanidine and � -lipoic acid, amino-guanidine, � -lipoic acid, pregnenolone, and coenzyme-Q 10 on the lifespan of mice. No treatment extended lifespan.

Received: February 22, 2007 Accepted: March 28, 2007 Published online: June 15, 2007

Stephen R. Spindler, PhD Department of Biochemistry, University of California 3401 Watkins Drive Riverside, CA 92521 (USA) Tel. +1 951 827 3597, Fax +1 951 827 4434, E-Mail spindler@ucr.edu

© 2007 S. Karger AG, Basel 0304–324X/07/0535–0306$23.50/0

Accessible online at: www.karger.com/ger

Screening Potential Longevity Therapeutics

Gerontology 2007;53:306–321 307

tive therapeutics can be developed. However, drug discovery and development have a long and successful history of using rapid, surrogate assays for identifying candidate therapeutics. Most of the medicants currently used to treat human diseases were initially discovered us-ing such surrogate assays, often without knowledge of the molecular mechanisms of the disease or the therapeutic. Thus, validated, surrogate assays may greatly enhance our ability to identify and develop longevity therapeu-tics.

Long-term CR (LTCR), undernutrition without mal-nutrition, usually started shortly after weaning, is a highly effective means of extending lifespan and reduc-ing the incidence and increasing the mean age of onset of many age-related diseases, including cancer, diabetes, renal failure, and some forms of neurodegeneration [1] . Disrupted insulin/insulin-like growth factor I (IGFI) signaling produces similar physiological and lifespan ef-fects, and in combination with LTCR, it can additively extend mammalian lifespan [2, 3] . Other long-lived mouse mutants also have been identified, suggesting that other, potentially druggable pathways may exist for ex-tending lifespan and ameliorating age-related diseases [4, 5] .

LTCR is usually begun soon after weaning in mam-mals. When CR is initiated in older B6C3F1 mice, it be-gins to extend their health and lifespan within 8 weeks [6, 7] . This extension of lifespan results primarily from reduced tumor-related mortality, especially reduced mortality from hepatocellular carcinoma (HCC), the ma-jor cause of mortality in this strain of mice. During the 8 weeks required to reduce mortality, 71% of the gene ex-pression changes induced by LTCR are reproduced in the livers of these mice ( table 1 ) [6–8] . When old LTCR mice are shifted to a control diet, almost all of the LTCR-re-lated gene expression reverts to control levels within 8 weeks [7, 9] . Similar results are found with heart gene ex-pression, although a smaller proportion of the LTCR re-sponsive genes are affected [9] . Thus, the physiological effects of CR appear to be closely linked to the changes it induces in global patterns of gene expression. This close linkage suggests that these changes can be used for screen-ing potential therapeutics for their CR-like effects on mammalian physiology. Such assays should avoid the ex-pense, time, and theoretical problems associated with lifespan assays in model organisms (see below). To test this hypothesis, we evaluated 4 potential therapeutics for their ability to reproduce the effects of LTCR in the liver of mice [10, 11] . We identified metformin as one such compound.

Are Lifespan Studies the Best Way to Search for Longevity Therapeutics?

The development of assays for longevity pharmaceu-ticals is problematic. Screening using short-lived organ-isms such as nematodes and flies is of unknown efficacy, and the results of such screening can be inconsistent [12–14] . Among the difficulties of such assays is the quantification of the calories consumed by each indi-vidual in the study. Historically, rodent lifespan studies have been the accepted method for screening potential longevity therapeutics. The NIA Interventions Testing Program is using this approach currently [15, 16] . The NIA began this effort after decades of limited success searching for physiological biomarkers capable of rap-idly detecting the underlying rate of aging. However, mouse lifespan studies suffer from limitations as well, many of which are illustrated by the study shown in fig-ure 1 .

We used cohorts of 60 B6C3F1 mice, a long-lived F1 hybrid strain, to assay the effects of 6 dietary supple-ments on lifespan. The mice were shifted to chemically defined diets and received either no supplements (con-trol) or melatonin; melatonin and pregnenolone; amino-guanidine; aminoguanidine and � -lipoic acid; or amino-guanidine, � -lipoic acid, pregnenolone, and coenzyme-Q 10 ( fig. 1 ). None of the compounds produced a statistically significant effect on lifespan. These data might be interpreted as evidence that the compounds have no affect on health or lifespan, at least in mice. However, most rodents die of a few characteristic dis-eases. In mice, these are often lymphoma or hepatoma [6, 7, 17] . Even the 4-way-cross mouse used by NIA Inter-ventions Testing Program suffers from this limitation [15, 16] . Between 48 and 80% of these mice die of tumors, although they die from a somewhat wider array of tu-mors than do most laboratory mice. 72–75% of the B6C3F1 mice we used die of hepatomas [7] . Thus, the lifespan studies described above would detect only sup-plements which intervene in spontaneous tumorigene-sis . If one or more of the supplements improved aging in another organ, such as brain or heart, this effect would not be detected. Further, the study required 300 singly caged mice, and lasted for 45 months. It would have last-ed even longer if one of the treatments had extended lifespan. Thus, this type of screening is time consuming, expensive, and limited in its scope. Finally, the use of shorter-lived, enfeebled rodent strains would introduce further confounds into the studies.

Spindler/Mote

Gerontology 2007;53:306–321 308

What Are We Measuring when We Perform Lifespan Studies?

Some might argue that such lifespan assays should detect the action of compounds that slow the underlying rate of aging, and that this action would slow the progress of most or all age-related diseases. Gerontologists often conjecture that CR slows the underlying rate of aging be-cause it can increase maximum lifespan in some organ-isms, including mice. However, CR can extend the maxi-

mum lifespan of mice by decreasing the rate of tumor growth [ 6, 7 ; and Spindler, unpubl. results]. Whether de-celerated tumor growth is rightly regarded as decelerated aging is open to question. But, few cancer chemothera-peutics would be regarded as longevity pharmaceuticals when administered to healthy subjects. Thus, the concept of underlying rate of aging, which has never been well defined, may have little useful meaning.

In fact, the effects of CR on cancer mortality may be incidental to its role in evolution. Widely accepted evolu-tionary theory holds that CR evolved early in metazoans as an adaptation to boom and bust cycles in the food sup-ply [18] . Since selection acts on reproductively active members of a population, it is difficult to rationalize the potent anticancer effects of CR with this theory. Cancer rates are low during the reproductive period of most mam-mals, and few individuals live long enough to die of cancer in the wild [19] . Interestingly, CR can reduce the growth rate of a mutationally induced tumor in nematodes, even though nematodes are largely postmitotic and do not nor-mally die of cancer [20] . Thus, the ancient pathways con-trolling longevity in metazoans may oppose neoplasms even in organisms not subject to them.

These considerations suggest that the anticancer ef-fects of CR arose by co-selection for another trait. We have proposed that this trait is the role played by cells as reser-voirs of metabolic energy [1] . Our high-density microar-ray studies identified a set of genes that respond similarly to CR in heart and liver [1] . The functions of these genes are consistent with the general, CR-related reduction in protein, RNA, lipid, and DNA synthesis reported in LTCR rodents [8, 21–23] . The tissue-specific changes in gene ex-pression we found are consistent with increased cell death and replacement in the mitotic liver, and with increased protein, nucleic acid, and lipid turnover in the postmi-totic heart [1] . These effects may lead to many of the anti-cancer and health benefits of CR.

Most cancers are derived from mitotically competent cells [24] . Cell division is required to genetically fix onco-genic mutations. While mitotic and postmitotic cells de-grade cellular protein and lipid for energy, some mitotic tissues, including liver and lung, undergo a profound, rap-id, and reversible loss of cell number (via apoptosis, necro-sis, and autophagic cell death) [25, 26] . This cell death preferentially affects preneoplastic and neoplastic cells [25] . Thus, the longevity effects of CR in mice may be de-rived in part from cell death in mitotically competent tis-sues, and protein turnover and cellular repair in both mi-totic and postmitotic tissues [1] . In this state, feeding in-duces a period of intense resynthesis and cell division (in

20

40

60

80

100

Months

Pe

rce

nt

surv

iva

l

15 20 25 30 35 40 45

0

Fig. 1. Longevity of mice treated with dietary supplements. A co-hort of mouse chow fed, male B6C3F1 mice (Harlan) were divided into groups of 60 mice when they were 14 months of age. Thereaf-ter, they were fed either control diet alone ( + ; AIN-93M; 84 kcal/week), or control diet with melatonin ( _ ; 41 � g/kg body weight/day), melatonin and pregnenolone ( o ; 41 and 200 � g/kg body weight/day, respectively), aminoguanidine ( p ; 65 mg/kg body weight), aminoguanidine and � -lipoic acid ( X ; 65 and 73 mg/kg body weight), or aminoguanidine, � - lipoic acid, pregnenolone, and coenzyme-Q 10 ( y ; 65, 73, 0.2, and 12 mg/kg body weight). Mice were fed three times weekly, and all food was eaten. Except for melatonin, supplements were mixed with the powered diet and cold-pressed into 1-gram pellets (BIO-SERV, Frenchtown, N.J., USA). The food was stored at 4 ° C until used. Melatonin was ad-ministered in acidified tap water (13.7 mg/l, adjusted to pH 4.0 with HCl) in brown water bottles from 5 p.m. to 8 a.m. The mice consumed approximately 2 ml of water during this time. From 8 a.m. to 6 p.m. the animals received the same acidified tap water as the other animals. All water bottles were changed weekly. A 12-hour light/dark cycle was utilized. The percentage of mice remain-ing alive at the end of each month is plotted.

Screening Potential Longevity Therapeutics

Gerontology 2007;53:306–321 309

some mitotic tissues). Alternating cycles of degradation and resynthesis may drive self-renewal and the elimina-tion of neoplastic and preneoplastic cells.

Should We Wait for Mechanism-Based Drug Development?

What is the best way to identify longevity pharmaceu-ticals? One approach would be to wait until aging is com-pletely understood, allowing the development of mecha-nism-based drugs. Some mechanism-based therapeutics are being used to treat patients. While there have been successes, there have also been well-publicized failures. The vast majority of the therapeutics in use today were developed without a mechanistic understanding of either the disease targeted or the drug tested. For example, long before the mechanisms of neoplastic transformation were understood, there were effective chemotherapeutics.

The development of metformin as an antidiabetic is an-other example of such drug discovery [27] . Treatments for diabetes mellitus existed for years to centuries before there was any knowledge of their physiologic mechanisms of ac-tion. The French lilac, Galega officinalis , was used in me-dieval times to relieve the excessive urination accompany-ing diabetes mellitus. Centuries later, the active agentwas identified as isoamylene guanidine. Independently, through a fortuitous study involving mistaken assump-tions, guanidine infusion was found to lower blood glucose concentrations. In the 1950s, this led to the development of biguanides, including metformin and phenformin, which were less toxic than guanidine. Following its me-dicinal use in Europe for 20 years, metformin was finally approved for use in the USA in 1995. Although now off patent, it remains a key treatment and preventative for type 2 diabetes. The molecular mechanisms of metformin ac-tion are still unclear, although they appear to include sig-naling through AMP-activated protein kinase [28] .

Does CR Work in Humans?

Some have argued that any intervention, including CR, is unlikely to extend human lifespan [29] . Phelan and co-workers, Rose and Demetrius, and De Grey have all ar-gued that the life history of humans presents little selec-tive pressure for a robust CR response [30–32] . While the validity of these hypotheses can be debated, the efficacy of human CR is unlikely to be known soon. However, even if CR does not extend human lifespan, it remains likely

that CR mimetics will have applications to our health. Analysis of data from the Baltimore Longitudinal Study of Aging suggests that physiological biomarkers associ-ated with lifespan extension in monkeys and rodents also are associated with enhanced lifespan in humans [33] . The molecular-genetic processes leading to lifespan ex-tension in CR animals may be active in some fortunate humans, perhaps due to our genetic diversity.

CR produces changes in human physiology closely as-sociated with enhanced longevity in model organisms. The consumption of a diet reduced 20% in calories by Oki-nawan adults in the 1970s was accompanied by a 50% re-duction in the mortality rate of 60- to 64-year-olds, and a 30–40% reduction in the death rate from malignancy, and cerebral vascular and heart disease, compared with the rest of Japan [34] . Okinawans who consumed a Western-style diet had mortality and morbidity rates approaching those of the west. Another study, often termed the Vallejo Nursing Home Study, found that CR reduced time in the infirmary (123 vs. 219 days; p ! 0.001) and produced nu-merically fewer deaths during a 3-year period (6 versus 13, not significantly different) in healthy volunteers over 65 years of age [35, 36] . Recently, it has been postulated that this may have been a study of alternate day hypo- and hy-percaloric diets rather than continuous CR [37] . A longi-tudinal human CR study serendipitously conducted on 8 healthy, nonobese humans eating a low-calorie diet for 2 years, found physiologic, hematologic, hormonal, and bio-chemical changes which resemble those of CR in rodents and monkeys [38] . Significantly reduced risk factors for atherosclerosis and better diastolic heart function were found in adult humans who had been CR for an average of 6 years [39–41] . Further, exercise training, decreased adi-posity, low protein intake, and LTCR in humans are all associated with low levels of plasma growth factors and hormones (especially IGFI) [e.g. 3–5, 42 ]. Lower IGF-I levels are associated with reater longevity in mice, Dro-sophila and C. elegans. Higher levels of these hormones and growth factors are linked to increased risk of cancer [42] . Thus, CR appears to produce many effects in humans which recapitulate those produced in invertebrates, ro-dents, dogs, and nonhuman primates.

Longevity Pharmaceuticals Should Be Efficacious whether or Not CR Extends Human Lifespan

Leaving aside the quality of life issues associated with prolonged human CR, dieting still is probably not the ideal way for humans to enjoy its health and longevity

Spindler/Mote

Gerontology 2007;53:306–321 310

benefits. Human malnutrition is associated with short stature, late reproductive maturation, lower baseline go-nadal steroid production, suppressed ovarian function, impaired lactation, reduced fecundity, weakened im-mune function, lower basal metabolic rate, reduced body temperature, enhanced irritability, reduced social interactions, loss of male libido, and in a few cases, hord-ing behavior [43–51] . These afflictions may not be the result of low quality diets, since similar physiological symptoms have been reported among CR rodents [43, 44] . In some, but not in all, studies of the association between body mass index and mortality in humans, a low BMI is associated with increased mortality [52–56] . For example, in a study of 14,407 individuals between 50 and 75 years of age, a BMI below 25 was associated with a steady increase in the probability of death from all causes in both men and women, after controlling for confounding variables [56] . Interestingly, enhanced mortality was mostly due to an increase in infectious and organic diseases. These results appear consistent with the immune suppression often reported in CR ro-dents and malnourished humans. Thus, it is possible that CR may have cumulatively negative consequences for humans. This would not be surprising, since humans are not confined to barrier vivaria, as are animals in most CR studies.

However, it is highly likely that the health- and life-span effects of CR can be separated from its negative ef-fects. Co-selection of most of the responses to CR item-ized above is easily rationalized as evolutionarily adap-tive. Thus, there is no reason to assume that they all are mediated by a single signal transduction pathway. In-deed, LTCR and dwarfism each can extend the lifespan of mice alone, while together they have an additive effect on lifespan. Our microarray studies indicate that CR and dwarfism signal through both common and distinct pathways to produce their lifespan effects (see below). Thus, multiple signaling pathways appear to be involved in their extension of mammalian lifespan. This suggests that the positive and negative effects of CR and dwarfism on physiology are due to multiple pathways, and therefore may be separable.

Humans may not be the longest lived animals. A Mar-ion’s tortoise was reported to have died at more than 150 years of age [57, 58] . Bowhead whales also may live to be more than 100 years old in the wild. Since 1981, Inupiat villagers along the north coast of Alaska have found six harpoon points made of ivory and stone in the blubber of freshly killed bowhead whales [59] . These harpoon points reportedly have not been used by whalers since

the 1880s, suggesting the whales were at least 100 years old when killed. Aspartic acid racemization studies of 94 eye lens nuclei from 84 individual bowheads suggest that five males were 113, 136, 160, 174, and 213 years old [60, 61] . Thus, these species may be capable of extreme lon-gevity without the downsides associated with CR or dwarfism.

Adaptations resulting in extreme longevity might in-volve either qualitative or quantitative changes in gene expression. Small molecule therapeutics are being devel-oped for genetic diseases caused by alterations in the lev-el and the activity of specific proteins [62, 63] . For ex-ample, clioquinol is able to downregulate the expression of a mutant Huntington gene mitigating its pathology in a Huntington’s disease model [62] . In another example, the abnormal chromosome segregation and binucleation resulting from a point mutation in the LMNA gene, which is responsible for Hutchinson-Gilford progeria syndrome, can be largely rescued with small molecule farnesyltrans-ferase inhibitors [64] . Likewise, the genes responsible for extreme mammalian longevity may be susceptible to small molecule therapeutics. However, the first require-ment for developing such therapeutics is a rapid assay for identifying their effects. We have begun to develop and validate one such surrogate assay.

Like CR, Longevity Therapeutics Could Act Rapidly

Aging is usually assumed to result from the gradual accrual of essentially irreversible oxidative or other dam-age to macromolecules. In this context, CR is often viewed as preventing or slowing the accumulation of such dam-age, thereby slowing the process of aging [65] . The inter-pretation of most CR studies has been strongly influenced by this view. Most cross-sectional studies of mammalian aging have been interpreted as if they were performed longitudinally, e.g. see the discussion and studies in Van Remmen et al. [66] . LTCR is almost always assumed to have prevented the incremental accumulation of irrevers-ible damage. However, in many of these studies it is not clear whether CR reduced the rate of damage or increased the rate of repair.

Ideal therapeutics act rapidly. Most people do not wor-ry about the effects of aging or lifestyle until they begin to experience the diseases associated with them. A lon-gevity therapeutic only capable of decreasing the accu-mulation of irreversible damage would do little to im-prove the health and longevity of the elderly. This type of drug would have to be consumed from birth through old

Screening Potential Longevity Therapeutics

Gerontology 2007;53:306–321 311

age. Instead, an ideal longevity therapeutic would act rap-idly to reduce age-related morbidity and mortality in the elderly. To test the hypothesis that CR can act rapidly, we conducted genome wide expression analysis of very old, control-fed mice shifted to a CR diet for just 4 weeks [8] . Because most mice of the strain used for these studies die of liver tumors, we focused first on this organ. The liver also has a major role in drug metabolism, glucose homeo-stasis, insulin responsiveness, and IGFI synthesis. Each of these functions is thought to be important in health and lifespan. We found that 4 weeks of CR reproduced 55% of all of the gene-expression effects of LTCR in el-derly mice. For age-responsive genes, 4 weeks of CR re-produced � 70% of the effects of LTCR. These results sug-gested to us that CR could act rapidly and proactively to initiate its health and lifespan effects. Recently, the im-portance of regulated apoptotic and autophagic turnover of damaged cells in lifespan determination has begun to be better appreciated [1] .

CR Can Act Rapidly, Even Late in Life, to Initiate Lifespan Extension

The effect of CR on lifespan is proportional to its du-ration [67] . However, CR was thought to extend life span only when it was initiated in mice which were 12-months old or younger [68] . Studies in rats suggested that CR is much less effective in older animals [69, 70] . Our results, described above, led us to test the possibility that CR could rapidly initiate its lifespan effects. To test this hy-pothesis, control fed, male, B6C3F1 mice were shifted to CR at the beginning of the accelerated mortality phase of their lifespan, at 19 months of age ( fig. 2 ). CR began to extend lifespan and reduce tumor incidence within 8 weeks ( fig. 2 ) [7] . Linear regression and breakpoint anal-ysis revealed that CR induced a 3.1-fold decrease in mor-tality within 2 months [6, 7] . CR also induced a 42% in-crease in time to death, and a substantial increase in mean (4.7 months) and maximum (6.0 months) lifespan.

20 24 28 32 36 40 44

20

40

60

80

100

Age (months)

Pe

rce

nt

surv

iva

l

Restricted

Control

First breakpoint

Secondbreakpoint

Reduced tumor

growth rate

Control CR

Reduced

tumor incidence

Control CR

a

b

c

a b

c

Fig. 2. The effects of CR begun immediately before the onset of the accelerated mortality phase of the lifespan curve. a The lon-gevity of mice switched from control to CR at 19 months of age. The percentage of mice remaining alive at the end of each month is shown for CR mice ( y ) and control mice ( U ). Shown is the mor-tality trajectory of the mice at the beginning of the experiment (––––), the slope of the accelerated mortality phase of the survival curve for the control mice (– – –), the approximate mortality tra-jectory of the CR mice between the first and second breakpoints in the survival curve (· · · · ·), and the mortality trajectory after the

second breakpoint (– · – · –). b A theoretical survival curve assum-ing that mortality of the CR mice results from a reduced rate of tumor growth and a constant rate of tumor formation. The low-ercase letters designate different parts of the curve. c A theoretical curve describing the results expected if mortality in the CR group results from reduced rates of tumor formation and a constant rate of tumor growth. Figure adapted from Spindler [6] , with permis-sion. For further explanation of the theoretical curves shown in b and c , see Spindler [6] .

Spindler/Mote

Gerontology 2007;53:306–321 312

In our hands, CR begun just after weaning also extends the remaining lifespan of these mice by about 40%.

The shapes of the survival curves suggest that the onset of CR reduced the rate of tumor growth more than the rate of tumor onset ( fig. 2 ) [6] . Follow-up studies suggest that the initiation of CR in older mice decreases the growth rate of spontaneous hepatocellular carcinomas, perhaps by increasing the rate of apoptotic or autophagic cell death [Spindler, Higami and Shimokawa, unpubl. results].

The reason that later-life CR was effective in extending lifespan in our studies and not in the rat studies is not known. However, it may be related to species-specific dif-ferences in the susceptibility of mice and rats to late-life CR, or to subtleties in the animal husbandry used in the studies. Examples of such methodological effects are the importance of a phased introduction of CR for lifespan extension in older mice and of companionship and/or warmth for the longevity of some types of dwarf mice [68, 71] .

Demographic studies in Drosophila have shown that their mortality rate is rapidly responsive to dietary calories [72] . Shifting the flies from a control to a CR diet deceler-ates their short-term risk of death within two days. Like-wise, shifting from a CR to a control diet accelerates their short-term risk of death in 2 days. The similarities between these results and ours suggest that the rapidity and revers-ibility of CR may be phylogenetically conserved. This is consistent with most evolutionary theories of CR. Organ-isms must be able to adapt their physiology and behavior rapidly to changes in the environment during the boom and bust cycles common to the life histories of most spe-cies.

Gene Expression Biomarkers Are Closely Linked to the Physiological Actions of CR

Gene expression patterns may be indicators of biolog-ical status. For example, the expression patterns of pri-mary tumor cells may be useful for predicting outcomes such as chemosensitivity, metastases, and survival [73, 74] . To investigate the relationship between gene-expres-sion and the health and longevity effects of CR, we exam-ined the induction of LTCR-like gene expression profiles in old mice shifted from a control diet to CR. We found that 2, 4 and 8 weeks of CR, initiated in older mice, pro-gressively induces LTCR-like gene expression profile in the liver [6] . The major response pattern is shown in fig-ure 3 . Thus, during the period of time required to initiate the longevity and anticancer effects of CR, the gene-ex-

pression profile of LTCR was substantially reproduced in liver. A similarly rapid response was found in heart, al-though fewer LTCR-responsive genes changed expres-sion in 8 weeks [9] . Nevertheless, the rapidly responsive genes appear to be key for reducing blood pressure, fibro-sis, and tissue remodeling, and for increasing cardiac contractility [9] . CR did not enhance apoptosis-related gene expression in heart, as it did in liver [1, 9] . Interest-ingly, in both heart and liver, the vast majority of the LTCR-responsive genes returned to control expression within levels 8 weeks of shifting to a control diet. Thus, essentially all LTCR-responsive genes are rapidly respon-sive to caloric intake.

CR Is Proactive rather than Protective

Our results are most consistent with the idea that CR rapidly and proactively alters gene expression and physi-ology to extend lifespan and improve health. Therefore, its major effect is unlikely to be preventing irreversible molecular damage. This view is consistent with the de-mographic studies in Drosophila discussed above [72] .

CR2 CON8

2194genes

Ge

ne

exp

ress

ion

Decrease

Control

level

Increase

14

7

1

14

5

3

10

17

21

1

11

2

5

14

21

10

1

2

4

14

7

1

5

10

17

15

CR4 CR8 LTCR

Fig. 3. Dynamics of the gene expression response in old control mice shifted to CR for 2 weeks (CR2), 4 weeks (CR4), and 8 weeks (CR8), and in old long-term CR (LTCR) mice shifted to control feeding for 8 weeks (CON8). The most common pattern of gene expression found is shown. Adapted from Dhahbi et a. [7] , with permission.

Screening Potential Longevity Therapeutics

Gerontology 2007;53:306–321 313

Biochemical and physiological studies conducted in ro-dents and humans also support this view. There is a small but growing literature regarding the effects of short-term CR on mammalian physiology and biochemistry. For ex-ample, just 1–3 months of food restriction can signifi-cantly increase the latency and reduce the incidence of spontaneous cancer over the entire lifespan of a mouse [75] . Just 1 week of CR induces apoptosis of the glutathi-one S-transferase-II positive (an immunohistochemical marker of preneoplastic liver cells) hepatocytes of old mice [76] . Forty-percent food restriction for 3 months eliminates 20–30% of liver cells through apoptosis, and reduces the number and volume of chemically induced preneoplastic foci by 85% [77] . In AD-transgenic mice, 6 and 14 weeks of CR substantially decreases the accumula-tion of A � plaques and astrocytic activation [78, 79] . Six weeks of CR in the old mice significantly reduces whole brain carbonyl and cortex sulfhydryl content (oxidative damage), although the effects are not as great as those of LTCR [80] . Similarly, 3–6 weeks of ad libitum feeding fully or partially reverses the effects of LTCR on the car-bonyl and sulfhydryl content of whole brain and heart [80] . In humans, short-term CR rapidly restores insulin sensitivity and lowers blood glucose levels in type 2 dia-betics [81, 82] . Moderate sustained weight loss from a CR diet can increase life expectancy and produce beneficial physiological changes related to diabetes, hypertension, hyperlipidemia, sleep apnea, and cardiorespiratory and other chronic degenerative diseases [83] . Short-term CR improves disease-related biomarkers associated with ag-ing in old, male rhesus monkeys [84] . Together, these re-sults suggest that CR can act rapidly to produce a physi-ological state associated with health and longevity in mammals, including humans.

Microarray-Derived Gene-Expression Patterns Can Be Used as Surrogate Biomarkers for Drug Discovery

The close temporal linkage between the health, lon-gevity and gene expression led us to hypothesize that the gene-expression changes induced by CR could be used as surrogates in drug discovery. To test this hypothesis, we evaluated the ability of 5 potential CR mimetics to repro-duce the gene-expression profiles associated with CR in mouse liver using Affymetrix microarrays [11] . We fo-cused the first studies on liver for the reasons given above. The linkage between CR, insulin, IGFI and the rate of ag-ing [4, 85] suggested that the glucoregulatory compounds metformin (MET), glipizide (GLIP), GLIP plus MET

(GM), and rosiglitazone (ROS) might be candidate CR mimetics. MET, a biguanide, increases insulin sensitivity in liver, muscle and adipose, and decreases hepatic glu-cose production and output [86] . ROS, a thiazolidinedi-one, is a peroxisome proliferator activated receptor (PPAR) gamma agonist that may improve insulin sensi-tivity in muscle and liver by interfering with the expres-sion and release of mediators of insulin resistance origi-nating in adipose tissue, such as ceramides, free fatty ac-ids, and adipocytokines [87] . GLIP, a sulfonylurea, is an insulin secretagogue in pancreatic � cells [88] . We also administered a combination of MET and GLIP, MG, be-cause glipizide enhanced insulin release might antago-nize the insulin-reducing effects of metformin on gene expression. Because of the importance of tumorigenesis in determining the lifespan of the mice, we also tested a putative chemopreventative, soy isoflavone extract (SOY), for its effects [89] . Soy isoflavones may act through vari-ous promiscuous nuclear receptors including the estro-gen, thyroid and PPARs [89] . One microarray study sug-gests that 19% of the effects of CR on gene expression may result from activation of signaling through the PPAR � receptor [90] .

MET, GLIP, GM, ROS or SOY were administered in the diets of mice for 8 weeks [11] . A control group re-ceived the diet free of drugs. All these groups were isoca-loric, and their weights did not vary significantly during the studies. Old control groups were included, which were either LTCR or control mice shifted to CR for 8 weeks (CR8). MET reproduced 75% of the gene expres-sion changes produced by LTCR. Eight weeks of CR re-produced only 71% effects of LTCR ( table 1 ). Eight weeks of MET treatment reproduced 74% of the gene expression effects of CR8 ( table 1 ). The other treatments were less effective. Thus, MET best replicates the gene-expression effects of LTCR.

The program Venn Mapper was used to investigate the similarities at increasing levels of statistical strin-gency [91] . MET and CR8 again yielded the highest number of genes overlapping those of LTCR ( fig. 4 ). We also determined the number of LTCR-like changes at increasing fold-change thresholds. MET, followed by CR8, again overlapped the most with LTCR [11] . These results indicate that MET reproduces the effects of LTCR even better than CR8, while the other treatments are less effective.

We also used GenMAPP and MAPPFinder to deter-mine the number of gene ontology (GO) terms common to LTCR and each treatment [11] . The number of overlap-ping terms is a nonbiased, quantitative measure of their

Spindler/Mote

Gerontology 2007;53:306–321 314

functional similarity [92, 93] . MET produced the highest number of overlapping GO terms, outstripping even the number produced by CR8 [11] . Thus, 8 weeks of MET treatment surpassed even 8 weeks of CR at producing a LTCR-like gene expression profile. These results suggest that MET may have significant LTCR-like effects on physiology and health. Based on these data, MET is a can-didate CR mimetic.

Some Physiological Effects of MET Are Consistent with Its Gene-Expression Profile

Consistent with the results reviewed above, MET treatment of female HER-2/neu mice increased their mean and maximum life spans by 8 and 13%. Also, MET reduced the incidence and size of mammary adenocar-cinomas and increased the latency of the tumors. A low-er dosage of MET prolonged the survival of a transgenic mouse model of Huntington’s disease by 20% [94] . Phen-formin, a biguanide which is structurally and function-ally related to metformin, extended the lifespan of C3H mice 23% while reducing tumor incidence by 80% [95, 96] . In humans with type 2 diabetes, MET may be asso-ciated with reduced cancer risk [97] . MET also protects hamsters fed a high-fat diet from malignant, hyperplas-tic and premalignant pancreatic lesions [98] . Indirect evidence suggests that signaling through AMP-activat-ed protein kinase could be involved in both the antidia-betic and the anticancer effects of MET [99] . AMP-acti-vated protein kinase signaling is capable of extending

lifespan in Caenorhabditis elegans , Drosophila and yeast [100–102] . MET treatment is also effective against poly-cystic ovary syndrome [103] . MET therapy also has been shown to inhibit the development of metabolic syn-drome in humans [104] . Metabolic syndrome is associ-ated with increased cardiovascular- and diabetes-asso-ciated morbidity and mortality. Together, these results suggest that use of microarray biomarkers has identified a promising candidate CR mimetic. However, it remains to be seen whether long-term treatment of healthy, long-lived mice with metformin will extend lifespan. Even

Table 1. Numerical overlap between the transcriptional effects of LTCR, CR8 and each of the drug treatments

Treatmentgroup

Number of LTCR-likeresponses*, %

Number of CR8-likeresponses*, %

CR8 71 –MET 75 74GLIP 16 17GM 20 23ROS 17 13SOY 11 13

* Number of LTCR- or CR8-responsive gene-expression changes produced by each treatment. MET = Metformin; GLIP = glipizide; GM = the combined administration of GLIP and MET; ROS = rosiglitazone; SOY = soy isoflavone extract. The percent-age of changed genes identical to those changed by LTCR or CR8 are shown. This table is adapted from Dhahbi et al. [11], with per-mission.

0

350 MET

CR8

ROS

GM

Nu

mb

er

of

ge

ne

s>2

50

100

150

200

250

300

GLIP

SOY

– >10 >15 >20 >22– – – –

Values

Fig. 4. The number of LTCR-like genes in-duced by each treatment at increasing lev-els of statistical stringency. Affymetrix data were filtered and normalized using MAS 5.0 and RMA, and subjected to multi-class SAM analysis followed by t tests to determine the effects of each dietary or drug treatment. The differentially ex-pressed genes were merged and analyzed using Venn Mapper to identify genes sig-nificantly affected by LTCR and each of the treatments. The comparisons were performed at a fold-change cut-off of 1.2 and the indicated z values. From Dhahbi et al. [11] , with permission.

Screening Potential Longevity Therapeutics

Gerontology 2007;53:306–321 315

low level toxicity, which might not be recognized during the drug approval process, might limit the efficacy of a drug taken over the course of a lifetime. Therefore it is important that we understand the entire gene-expres-sion profile produced by a drug or treatment, so that we can more accurately predict its health effects.

Gene-Expression Biomarkers of Longevity

To broaden our understanding of longevity-associated gene expression, we investigated the hepatic gene-expres-sion patterns associated with CR and dwarfism [2] . LTCR and dwarfism together had additive effects on the expres-sion of 100 genes ( fig. 5 a). For example, if CR and dwarf-ism each induced the expression of a gene by 2-fold, then together they induced its expression by 4-fold. Dwarfism also affected the expression of 212 other genes, whether or not CR was present ( fig. 5 a). Likewise, CR affected the ex-pression of 77 genes, whether or not dwarfism was pres-

ent. One of a number of possible mechanisms by which such effects could be mediated at the molecular level by two signaling systems is shown in figure 5 b.

CR and dwarfism produced changes in gene expres-sion consistent with increased insulin, glucagon and cat-echolamine sensitivity. We found additive effects of CR and dwarfism on genes associated with apoptosis, gly-colysis, signal transduction, translation, RNA splicing, chaperones, transcription and xenobiotic metablism ( fig. 6 ). Both treatments also strongly downregulated genes associated with cholesterol, fatty acid, and lipid biosynthesis, and immune function ( fig. 6 ). However, there was no evidence for additivity in these effects. In-stead, they often affected the expression of different genes in the same pathways. Together, these sets of genes, par-ticularly those additively affected should prove useful for identifying potential longevity therapeutics.

A number of other genetic mouse models have been reported to enhance longevity, and these models may provide additional biomarkers for drug discovery [4, 5,

Treatment Dwarfism LTCR

212 genesGene expression100 additive or

interacting genes77 genes

Lifespan extension by DF only

Lifespan extension by CR only

Lifespan extension by CR and DF together

Phenotype

a

b Dwarfism LTCR

Individual effects

Independent-

additive effects

Interaction between

diet and genotype

Gene 1 Gene 2

Gene 3

Gene 4

Fig. 5. A summary of hepatic gene expres-sion profiling of normal and dwarf mice fed ad libitum or LTCR. a Dwarfism changed the expression of 312 genes (212 + 100 genes), LTCR changed the expression of 177 genes (77 + 100), and dwarfism and LTCR together changed the expression of 389 genes (212 + 100 + 77 genes). Of the 100 additively changed genes, 95 showed no statistical evidence of an interaction be-tween dwarfism and CR, while 5 showed evidence of an interaction. b A model for the regulation of 212 genes by dwarfism (hypothetical gene 1), 77 genes by CR (hy-pothetical gene 2), 95 genes additively by CR and dwarfism (hypothetical gene 3), and 5 genes interactively by CR and dwarf-ism (hypothetical gene 4). The double-headed arrow indicates a physical or func-tional interaction between transcription factors bound to adjacent sites which syn-ergistically alters their activity. Adapted from Tsuchiya et al. [2], with permission.

Spindler/Mote

Gerontology 2007;53:306–321 316

105] . For example, overexpression of the Klotho gene product, which binds to a cell surface receptor to suppress action of the insulin/IGFI signaling pathway, can extend mouse lifespan by 20–30% [4] . Microarray analysis of these mutants may provide additional gene expression biomarkers for drug discovery. Dietary manipulations other than CR, such as methionine or tryptophan restric-tion, or every-other-day feeding, may also provide useful biomarkers [106–112] .

Distinguishing the Beneficial Effects of CR from Its Negative Effects

One important aspect of drug screening, alluded to above, is the ability to distinguish positive from negative physiological outcomes. As a model for interpreting gene expression profiles, we reasoned that low insulin diabetes might produce a gene expression signature partially over-lapping that of LTCR. Forty percent LTCR in mice is characterized by a 66% reduction in fasting insulin levels [113] . To explore the relationship between CR and low-insulin diabetes, we profiled the effect of streptozotocin-induced diabetes (SID) on mRNA expression in mouse liver [114] . We found that SID, like LTCR, enhances the expression of hepatic genes associated with protein deg-radation and apoptosis. However, while LTCR enhanced

transcript levels associated with cell and protein renewal, SID altered gene expression in a manner consistent with reduced cell and protein renewal. These results empha-size the importance of the combinatorial effects of genes, and not simply the over- or underexpression of individu-al genes, to a physiological outcome.

How Does Altered Insulin and IGFI Signaling Enhance Lifespan?

In the mouse, as we show above, lifespan studies are primarily studies of the effects of treatments on tumor-igenesis, frequently HCC. Insulin and/or IGFI signaling are reduced in most or all models of extended longevity, including methionine restriction and Klotho overex-pression [5, 10, 106, 115] . In many tumors, including HCC, the level of expression of the type I IGFI receptor (IGFR) has been correlated with disease stage, reduced survival, development of metastases, and tumor de-dif-ferentiation [116–118] . Reduction of insulin/IGFI sig-naling in mice by mutation, genetic manipulation, or Klotho overexpression enhances the rate of tumor cell apoptosis, reduces the number and the volume of pre-neoplastic lesions in the liver, delays tumor-associated mortality, and extends lifespan [115, 119] . A part of the antineoplastic effects of CR in the liver and other tissues

Dwarf

F

Transcription

Cholesterol synthesis

Fatty acid synthesis

Lipid transport

Immune system

Cell cycle

DNA replication

Nucleotide

metabolism

structure

Treatment CR

212 genesinteracting) genes

77 Genes

F Apoptosis

F Glycolysis

Signal transduction

Translation

RNA splicing

F Chaperone

F Protein turnover

F Oxidant metabolism

F Cholesterol synthesis

F Fatty acid synthesis

F Lipid transport

F Immune system

F Gluconeogenesis

F Beta-oxidation

Histone modulation

F

F

F Protein turnover

F Oxidant metabolism

Xenobiotic

metabolism

F

F

F

F

F

F

F

F

Cell cycle

F

DNA replication

F

Nucleotide

metabolism

Transport and trafficking

Cell adhesion and

structure

Treatment

Gene expression100 additive (or

77 genes

Gene ontology

F Apoptosis

F

Glycolysis

Signal transduction

Translation

RNA splicing

F

Chaperone

F Protein turnoverF Oxidant metabolism

F

Cholesterol synthesis

F

Fatty acid synthesis

F

Lipid transport

F

Immune system

F Gluconeogenesis

F

Beta-oxidation

Histone modulation

F Apoptosis

Glycolysis

Signal transduction

Translation

RNA splicing

F

Chaperone

F

F

Transcription

F Xenobiotic

metabolism

Apoptosis

Glycolysis

Signal transduction

Translation

RNA splicing

Chaperone

Fig. 6. Cellular processes responsive to dwarfism and CR in mice. Gene ontology classifications of regulated genes were de-termined manually by examination of the PubMed, GenBank, NCBI, Gene Cards, NetAffx, EMBL Bioinformatic Harvester, LocusLink, and MGI online databases. The consensus functional pathways and biological processes associated with each gene was judged by examination of the rel-evant literature. Pathways and processes shared by dwarfism and CR are shown in italics. Up and down arrows indicate that for that process or pathway most or all genes were increased or decreased, respec-tively, by the indicated treatment. The ab-sence of an arrow indicates there was a mixed response in that process or path-way.

Screening Potential Longevity Therapeutics

Gerontology 2007;53:306–321 317

are probably the direct result of reduced IGFR signaling [120, 121] . IGFR is present at very low levels in normal hepatocytes [122] . Cells that do not express IGFR are resistant to transformation by any means [123] . How-ever, significant increases in insulin-like growth factor II (IGFII) and IGFR expression are found in human cir-rhotic liver (a preneoplastic state) and in some primary liver cancers versus normal adult liver [122, 124–127] . IGFII gene expression is activated during hepatocar-cinogenesis in some transgenic mice [128] . IGFR ex-pression is also upregulated in hepatoma cell lines de-rived from humans [129–133] , chickens [134] , and rats [135] . In several human HCC cell lines, blockade of IGFR with the selective inhibitor NVP-AEW541 in-duced growth inhibition, apoptosis, and cell cycle arrest [121] . Signaling of both IGFI and IGFII through the IGFR is associated with hepatocarcinogenesis and resis-tance to drug-induced apoptosis [136] . In hepatitis B-infected liver and hepatoma cells, expression of the viral HBx protein, which is important to HBV-associated car-cinogenesis, appears to induce the expression of IGFII and IGFR [137–139] . In such cells, elevated IGFR expres-sion appears to increase the mitogenic effect of IGFI and IGFII [138, 139] . While IGFI and IGFII are synthesized by hepatocytes, IGFII is also synthesized by resident macrophages, endothelial cells and stellate cells [140] . These studies suggest an autocrine or paracrine role for IGFI and IGFII in HCC growth and resistance to apop-tosis.

According to the classical view, the insulin receptor (IR) predominantly mediates anabolic effects, while the IGFR predominantly mediates antiapoptotic, mitogenic, and transforming effects [123, 141] . However, insulin can be a strongly antiapoptotic, co-mitogen in the liver and other tissues [142] . Several lines of evidence support a mitogenic and transforming role for the IRA splice vari-ant of the insulin receptor in hepatocarcinogenesis. Both IRA and IRB (IRB is the IR isoform normally found in insulin-responsive tissues) are expressed in hepatocytes and hepatoma cells [143, 143] . Both IGFI and IGFII are high-affinity ligands and activators of IRA. Activation of IRA can promote growth and protect malignant cells from apoptosis [144–146] . Binding of IGFII or insulin to IRA induces gene expression profiles which only partial-ly overlap those of IRB [147, 148] . In cells expressing only IRA, IGFII stimulates mainly the Shc/ERK branch of the insulin/growth factor signaling pathway, inducing mito-genesis and migration more potently than insulin [149] . In contrast, insulin is more potent than IGFII in stimu-lating signaling through the IRS/AKT pathway, leading

to enhanced glucose uptake and metabolism, protein synthesis and cell growth [149] .

Thus, it is highly likely that reduced insulin and IGFI signaling enhance apoptosis and reduce tumor cell growth and division in mice. These effects may be key to the anticancer and longevity effects of CR and the other means of lifespan extension in mice and possibly other organisms.

Conclusions

CR begun relatively late in the lifespan of mice rap-idly begins to decelerate mortality, extend remaining lifespan, and delay the onset and/or progression of can-cer as a cause of death. These health and longevity ef-fects coincide with a LTCR-specific gene-expression profile in the liver. This profile appears to be causally linked to the physiological effects of CR. We have used a LTCR-related profile as a biomarker to identify poten-tial CR mimetics. Metformin was one such potential mi-metic. Distinct patterns of gene expression induced by dwarfism and low insulin diabetes indicate that the combinatorial effects of many genes are important in reproducing the health- and lifespan extending effects of CR. These results suggest that longevity enhancing medicaments can be developed or discovered using such gene-expression biomarkers. It will be important to de-termine whether candidate longevity therapeutics can extend the lifespan of healthy individuals when taken over a lifetime.

Acknowledgements

The authors thanks their laboratory colleagues, cited in our articles above, for their hard work and many helpful discus-sions.

Spindler/Mote

Gerontology 2007;53:306–321 318

References

1 Spindler SR, Dhahbi JM: Conserved and tis-sue-specific genic and physiological re-sponses to caloric restriction and altered IGFI signaling in mitotic and postmitotic tissues. Annu Rev Nutr 2007;27:193–217.

2 Tsuchiya T, Dhahbi JM, Cui X, Mote PL, Bartke A, Spindler SR: Additive regulation of hepatic gene expression by dwarfism and ca-loric restriction. Physiol Genomics 2004; 17: 307–315.

3 Bartke A, Wright JC, Mattison JA, Ingram DK, Miller RA, Roth GS: Extending the life-span of long-lived mice. Nature 2001; 414: 412.

4 Kurosu H, Yamamoto M, Clark JD, Pastor JV, Nandi A, Gurnani P, McGuinness OP, Chikuda H, Yamaguchi M, Kawaguchi H, Shimomura I, Takayama Y, Herz J, Kahn CR, Rosenblatt KP, Kuro O: Suppression of aging in mice by the hormone Klotho. Science 2005; 309: 1829–1833.

5 Liang H, Masoro EJ, Nelson JF, Strong R, Mc-Mahan CA, Richardson A: Genetic mouse models of extended lifespan. Exp Gerontol 2003; 38: 1353–1364.

6 Spindler SR: Rapid and reversible induction of the longevity, anticancer and genomic ef-fects of caloric restriction. Mech Age Dev 2005; 126: 960–966.

7 Dhahbi JM, Kim HJ, Mote PL, Beaver RJ, Spindler SR: Temporal linkage between the phenotypic and genomic responses to calor-ic restriction. Proc Natl Acad Sci USA 2004; 101: 5524–5529.

8 Cao SX, Dhahbi JM, Mote PL, Spindler SR: Genomic profiling of short- and long-term caloric restriction effects in the liver of aging mice. Proc Natl Acad Sci USA 2001; 98: 10630–10635.

9 Dhahbi JM, Tsuchiya T, Kim HJ, Mote PL, Spindler SR: Gene expression and physiolog-ic responses of the heart to the initiation and withdrawal of caloric restriction. J Gerontol [A] 2006; 61: 218–231.

10 Spindler SR: Use of microarray biomarkers to identify longevity therapeutics. Aging Cell 2006; 5: 39–50.

11 Dhahbi JM, Mote PL, Fahy GM, Spindler SR: Identification of potential caloric restriction mimetics by microarray profiling. Physiol Genomics 2005; 23: 343–350.

12 Bayne AC, Sohal RS: Effects of superoxide dismutase/catalase mimetics on life span and oxidative stress resistance in the house-fly, Musca domestica . Free Rad Biol Med 2002; 32: 1229–1234.

13 Melov S, Ravenscroft J, Malik S, Gill MS, Walker DW, Clayton PE, Wallace DC, Mal-froy B, Doctrow SR, Lithgow GJ: Extension of life-span with superoxide dismutase/cata-lase mimetics. Science 2000; 289: 1567–1569.

14 Keaney M, Matthijssens F, Sharpe M, Van-fleteren J, Gems D: Superoxide dismutase mimetics elevate superoxide dismutase ac-tivity in vivo but do not retard aging in the nematode Caenorhabditis elegans . Free Rad-ic Biol Med 2004; 37: 239–250.

15 Miller RA, Chrisp C: T cell subset patterns that predict resistance to spontaneous lym-phoma, mammary adenocarcinoma, and fi-brosarcoma in mice. J Immunol 2002; 169: 1619–1625.

16 Nadon NL: Exploiting the rodent model for studies on the pharmacology of lifespan ex-tension. Aging Cell 2006; 5: 9–15.

17 Turturro A, Duffy P, Hass B, Kodell R, Hart R: Survival characteristics and age-adjusted disease incidences in C57BL/6 mice fed a commonly used cereal-based diet modulated by dietary restriction. J Gerontol [A] 2002; 57:B379–B389.

18 Masoro EJ, Austad SN: The evolution of the antiaging action of dietary restriction: A hy-pothesis. J Gerontol [A] 1996; 51A:B387–B391.

19 Phelan JP, Austad SN: Natural selection, di-etary restriction, and extended longevity. Growth Dev Aging 1989; 53: 4–6.

20 Pinkston JM, Garigan D, Hansen M, Kenyon C: Mutations that increase the life span of C. elegans inhibit tumor growth. Science 2006; 313: 971–975.

21 Lewis SE, Goldspink DF, Phillips JG, Merry BJ, Holehan AM: The effects of aging and chronic dietary restriction on whole body growth and protein turnover in the rat. Exp Gerontol 1985; 20: 253–263.

22 Merry BJ, Holehan AM: In vivo DNA syn-thesis in the dietary restricted long-lived rat. Exp Gerontol 1985; 20: 15–28.

23 Chung HY, Sung B, Jung KJ, Zou Y, Yu BP: The molecular inflammatory process in ag-ing. Antioxid Redox Signal 2006; 8: 572–581.

24 Wright KM, Deshmukh M: Restricting apoptosis for postmitotic cell survival and its relevance to cancer. Cell Cycle 2006; 5: 1616–1620.

25 Kouda K, Nakamura H, Kohno H, Ha-Kawa SK, Tokunaga R, Sawada S: Dietary restric-tion: effects of short-term fasting on protein uptake and cell death/proliferation in the rat liver. Mech Age Dev 2004; 125: 375–380.

26 Massaro D, DeCarlo MG, Baras A, Hoffman EP, Clerch LB: Calorie-related rapid onset of alveolar loss, regeneration, and changes in mouse lung gene expression. Am J Physiol 2004; 286:L896–L906.

27 Witters LA: The blooming of the French li-lac. J Clin Invest 2001; 108: 1105–1107.

28 Zhou G, Myers R, Li Y, Chen Y, Shen X, Fe-nyk-Melody J, Wu M, Ventre J, Doebber T, Fujii N, Musi N, Hirshman MF, Goodyear LJ, Moller DE: Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest 2001; 108: 1167–1174.

29 Hayflick L: ‘Anti-aging’ is an oxymoron. J Gerontol [A] 2004; 59:B573–B578.

30 Phelan JP, Rose MR: Caloric restriction in-creases longevity substantially only when the reaction norm is steep. Biogerontology 2006; 7: 161–164.

31 Demetrius L: Of mice and men. When it comes to studying ageing and the means to slow it down, mice are not just small humans. EMBO Rep 2005; 6(Spec No):S39–S44.

32 De Grey AD: The unfortunate influence of the weather on the rate of ageing: why human caloric restriction or its emulation may only extend life expectancy by 2–3 years. Geron-tology 2005; 51: 73–82.

33 Roth GS, Lane MA, Ingram DK, Mattison JA, Elahi D, Tobin JD, Muller D, Metter EJ: Biomarkers of caloric restriction may predict longevity in humans. Science 2002; 297: 811.

34 Kagawa Y: Impact of Westernization on the nutrition of Japanese: changes in physique, cancer, longevity and centenarians. Prev Med 1978; 7: 205–217.

35 Stunkard AJ: Nutrition, aging and obesity; in Rockstein M, Sussman ML (eds): Nutrition, Longevity, and Aging. Proceedings of a Sym-posium on Nutrition, Longevity, and Aging, held in Miami, Florida, February 26–27, 1976. New York, Academic Press, 1976, pp 253–284.

36 Vallejo EA: Hunger diet on alternate days in the nutrition of the aged. Prensa Med Argent 1957; 44: 119–120.

37 Johnson JB, Laub DR, John S: The effect on health of alternate day calorie restriction: eating less and more than needed on alter-nate days prolongs life. Med Hypoth 2006; 67: 209–211.

38 Walford RL, Mock D, Verdery R, MacCal-lum T: Calorie restriction in biosphere 2: al-terations in physiologic, hematologic, hor-monal, and biochemical parameters in humans restricted for a 2-year period. J Gerontol [A] 2002; 57:B211–B224.

39 Fontana L, Meyer TE, Klein S, Holloszy JO: Long-term calorie restriction is highly effec-tive in reducing the risk for atherosclerosis in humans. Proc Natl Acad Sci USA 2004; 101: 6659–6663.

40 Meyer TE, Kovacs SJ, Ehsani AA, Klein S, Holloszy JO, Fontana L: Long-term caloric restriction ameliorates the decline in dia-stolic function in humans. J Am Coll Car-diol 2006; 47: 398–402.

41 Fontana L, Klein S: Aging, adiposity, and cal-orie restriction. JAMA 2007; 297: 986–994.

42 Fontana L, Klein S, Holloszy JO: Long-term low-protein, low-calorie diet and endurance exercise modulate metabolic factors associ-ated with cancer risk. Am J Clin Nutr 2006; 84: 1456–1462.

Screening Potential Longevity Therapeutics

Gerontology 2007;53:306–321 319

43 Smith JV, Heilbronn LK, Ravussin E: Energy restriction and aging. Curr Opin Clin Nutr Metab Care 2004; 7: 615–622.

44 Weindruch R, Walford RL: The Retardation of Aging and Disease by Dietary Restriction, ed 1. Springfield, Thomas, 1988.

45 Ellison PT: Age and developmental effects on adult ovarian function; in Rosetta L, Mas-cie-Taylor NCG (eds): Variability in Human Fertility: A Biological Anthropological Ap-proach. New York, Cambridge University Press, 1996, pp 69–90.

46 Prentice AM, Lunn PG, Watkinson M, Whitehead RG: Dietary supplementation of lactating Gambian women. II. Effect on ma-ternal health, nutritional status and bio-chemistry. Hum Nutr Clin Nutr 1983; 37: 65–74.

47 Lager C, Ellison PT: Effects of moderate weight loss on ovarian function assessed by salivary progesterone measurements. Am J Human Biol 1990; 2: 303–312.

48 Lee RB: The !Kung San: Men, Women, and Work in a Foraging Society. Chicago, Cam-bridge University Press, 1979.

49 Somes GW, Kritchevsky SB, Shorr RI, Pahor M, Applegate WB: Body mass index, weight change, and death in older adults: the sys-tolic hypertension in the elderly program. Am J Epidemiol 2002; 156: 132–138.

50 Pasanisi F, Contaldo F, de Simone G, Man-cini M: Benefits of sustained moderate weight loss in obesity. Nutr Metab Cardio-vasc Dis 2001; 11: 401–406.

51 Keys A, Brozek J, Henschel A, Mickelson O, Taylor H: The Biology of Human Starvation. Minneapolis, University of Minnesota Press, 1950, vol 2.

52 Ajani UA, Lotufo PA, Gaziano JM, Lee IM, Spelsberg A, Buring JE, Willett WC, Manson JE: Body mass index and mortality among US male physicians. Ann Epidemiol 2004; 14: 731–739.

53 Lindsted K, Tonstad S, Kuzma JW: Body mass index and patterns of mortality among Seventh-day Adventist men. Int J Obes 1991; 15: 397–406.

54 Okamoto Y, Miyazaki N, Kurumagawa H, Fujino K, Mori K, Shimizu S, Ishikawa K: Re-lationship between morbidity and body mass index of mariners in the Japan Maritime Self-Defense Force Fleet Escort Force. Mil Med 2001; 166: 681–684.

55 Manson JE, Willett WC, Stampfer MJ, Cold-itz GA, Hunter DJ, Hankinson SE, Hennek-ens CH, Speizer FE: Body weight and mor-tality among women. N Engl J Med 1995; 333: 677–685.

56 Sunder M: Toward generation XL: anthropo-metrics of longevity in late 20th-century United States. Econ Hum Biol 2005; 3: 271–295.

57 Flower SS: Further notes on the duration of life in animals. 3. Reptiles. Proc Zool Soc Lond [A] 1937; 107: 1–39.

58 Comfort A: The Biology of Senescence, ed 3. Edinburgh, Churchill-Livingstone, 1979.

59 Rogers P: The Old Men of the Sea: Whales Longevity. San Jose Mercury News 2000; 19 December.

60 George JC, Bada JL, Zeh J, Scott L, Brown SE, O’Hara T, Suydam R: Age and growth esti-mates of bowhead whales (Balaena mystice-tus) via aspartic acid racemization. Can J Zool 1999; 77: 571–580.

61 Rosa C, George JC, Zeh J, O’Hara TM, Botta O, Bada JL: Update on age estimation of bow-head whales (Balaena mysticutus) using as-partic acid racimization. Proceedings Inter-national Whaling Commission, 2005.

62 Nguyen T, Hamby A, Massa SM: Clioquinol down-regulates mutant Huntington expres-sion in vitro and mitigates pathology in a Huntington’s disease mouse model. Proc Natl Acad Sci USA 2005; 102: 11840–11845.

63 Gelb MH, Brunsveld L, Hrycyna CA, Mi-chaelis S, Tamanoi F, Van Voorhis WC, Waldmann H: Therapeutic intervention based on protein prenylation and associated modifications. Nat Chem Biol 2006; 2: 518–528.

64 Cao K, Capell BC, Erdos MR, Djabali K, Col-lins FS: A lamin A protein isoform overex-pressed in Hutchinson-Gilford progeria syn-drome interferes with mitosis in progeria and normal cells. Proc Natl Acad Sci USA 2007; 104: 4949–4954.

65 Bokov A, Chaudhuri A, Richardson A: The role of oxidative damage and stress in aging. Mech Age Dev 2004; 125: 811–826.

66 Van Remmen H, Ward WF, Sabia RV, Rich-ardson A: Gene expression and protein deg-radation; in Masoro EJ (ed): Handbook of Physiology. Section 11: Aging. New York, Oxford University Press, 1995, pp 171–234.

67 Merry BJ: Molecular mechanisms linking calorie restriction and longevity. Int J Bio-chem Cell Biol 2002; 34: 1340–1354.

68 Weindruch R, Walford RL: Dietary restric-tion in mice beginning at 1 year of age: effect on life-span and spontaneous cancer inci-dence. Science 1982; 215: 1415–1418.

69 Lipman RD, Bronson RT, Wu D, Smith DE, Prior R, Cao G, Han SN, Martin KR, Mey-dani SN, Meydani M: Disease incidence and longevity are unaltered by dietary antioxi-dant supplementation initiated during mid-dle age in C57BL/6 mice. Mech Age Dev 1998; 103: 269–284.

70 Lipman RD, Smith DE, Bronson RT, Blum-berg J: Is late-life caloric restriction benefi-cial? Aging (Milano) 1995; 7: 136–139.

71 Brown-Borg HM, Borg KE, Meliska CJ, Bartke A: Dwarf mice and the ageing pro-cess. Nature 1996; 384: 33.

72 Mair W, Goymer P, Pletcher SD, Partridge L: Demography of dietary restriction and death in Drosophila . Science 2003; 301: 1731–1733.

73 Vasselli JR, Shih JH, Iyengar SR, Maranchie J, Riss J, Worrell R, Torres-Cabala C, Tabios R, Mariotti A, Stearman R, Merino M, Wal-ther MM, Simon R, Klausner RD, Linehan WM: Predicting survival in patients with metastatic kidney cancer by gene-expression profiling in the primary tumor. Proc Natl Acad Sci USA 2003; 100: 6958–6963.

74 Rosenwald A, Wright G, Chan WC, et al: The use of molecular profiling to predict surviv-al after chemotherapy for diffuse large-B-cell lymphoma. N Engl J Med 2002; 346: 1937–1947.

75 Klebanov S: Can short-term dietary restric-tion and fasting have a long-term anticarci-nogenic effect? Interdiscip Top Gerontol 2007; 35: 176–192.

76 Muskhelishvili L, Turturro A, Hart RW, James SJ: Pi-class glutathione-S-transferase-positive hepatocytes in aging B6C3F1 mice undergo apoptosis induced by dietary re-striction. Am J Pathol 1996; 149: 1585–1591.

77 Grasl-Kraupp B, Bursch W, Ruttkay-Nedecky B, Wagner A, Lauer B, Schulte-Hermann R: Food restriction eliminates preneoplastic cells through apoptosis and antagonizes car-cinogenesis in rat liver. Proc Natl Acad Sci USA 1994; 91: 9995–9999.

78 Patel NV, Gordon MN, Connor KE, Good RA, Engelman RW, Mason J, Morgan DG, Morgan TE, Finch CE: Caloric restriction at-tenuates Abeta-deposition in Alzheimer transgenic models. Neurobiol Aging 2005; 26: 995–1000.

79 Wang J, Ho L, Qin W, Rocher AB, Seror I, Humala N, Maniar K, Dolios G, Wang R, Hof PR, Pasinetti GM: Caloric restriction attenu-ates beta-amyloid neuropathology in a mouse model of Alzheimer’s disease. FASEB J 2005; 19: 659–661.

80 Forster MJ, Sohal BH, Sohal RS: Reversible effects of long-term caloric restriction on protein oxidative damage. J Gerontol [A] 2000; 55:B522–B529.

81 Capstick F, Brooks BA, Burns CM, Zilkens RR, Steinbeck KS, Yue DK: Very low calorie diet (VLCD): a useful alternative in the treat-ment of the obese NIDDM patient. Diabetes Res Clin Pract 1997; 36: 105–111.

82 Halter JB: Carbohydrate metabolism; in Masoro EJ (ed): Handbook of Physiology. Section 11: Aging. New York, Oxford Uni-versity Press, 1995, pp 119–145.

83 Moderate weight loss: many health benefits. Mayo Clin Health Lett 2005; 23: 4.

84 Lane MA, Tilmont EM, De Angelis H, Han-dy A, Ingram DK, Kemnitz JW, Roth GS: Short-term calorie restriction improves dis-ease-related markers in older male rhesus monkeys (Macaca mulatta) . Mech Age Dev 2000; 112: 185–196.

85 Bartke A: Minireview: role of the growth hormone/insulin-like growth factor system in mammalian aging. Endocrinology 2005; 146: 3718–3723.

Spindler/Mote

Gerontology 2007;53:306–321 320

86 Radziuk J, Bailey CJ, Wiernsperger NF, Yud-kin JS: Metformin and its liver targets in the treatment of type 2 diabetes. Curr Drug Tar-gets Immune Endocr Metabol Disord 2003; 3: 151–169.

87 Stumvoll M, Haring HU: Glitazones: clinical effects and molecular mechanisms. Ann Med 2002; 34: 217–224.

88 Rendell M: The role of sulphonylureas in the management of type 2 diabetes mellitus. Drugs 2004; 64: 1339–1358.

89 Ricketts ML, Moore DD, Banz WJ, Mezei O, Shay NF: Molecular mechanisms of action of the soy isoflavones includes activation of promiscuous nuclear receptors: a review. J Nutr Biochem 2005; 16: 321–330.

90 Corton JC, Apte U, Anderson SP, Limaye P, Yoon L, Latendresse J, Dunn C, Everitt JI, Voss KA, Swanson C, Kimbrough C, Wong JS, Gill SS, Chandraratna RA, Kwak MK, Kensler TW, Stulnig TM, Steffensen KR, Gustafsson JA, Mehendale HM: Mimetics of caloric restriction include agonists of lipid-activated nuclear receptors. J Biol Chem 2004; 279: 46204–46212.

91 Smid M, Dorssers LC, Jenster G: Venn map-ping: clustering of heterologous microarray data based on the number of co-occurring differentially expressed genes. Bioinformat-ics 2003; 19: 2065–2071.

92 Dahlquist KD, Salomonis N, Vranizan K, Lawlor SC, Conklin BR: GenMAPP, a new tool for viewing and analyzing microarray data on biological pathways. Nat Genet 2002; 31: 19–20.

93 Doniger SW, Salomonis N, Dahlquist KD, Vranizan K, Lawlor SC, Conklin BR:MAPP Finder: using Gene Ontology and GenMAPP to create a global gene-expres-sion profile from microarray data. Genome Biol 2003; 4:R7.

94 Ma TC, Buescher JL, Oatis B, Funk JA, Nash AJ, Carrier RL, Hoyt KR: Metformin therapy in a transgenic mouse model of Huntington’s disease. Neurosci Lett 2007; 411: 98–103.

95 Anisimov VN, Semenchenko AV, Yashin AI: Insulin and longevity: antidiabetic bigua-nides as geroprotectors. Biogerontology 2003; 4: 297–307.

96 Dilman VM, Anisimov VN: Effect of treat-ment with phenformin, diphenylhydantoin or L -dopa on life span and tumour incidence in C3H/Sn mice. Gerontology 1980; 26: 241–246.

97 Evans JMM, Donnelly LA, Emslie-Smith AM, Alessi DR, Morris AD: Metformin and reduced risk of cancer in diabetic patients. BMJ 2005; 330: 1304–1305.

98 Schneider MB, Matsuzaki H, Haorah J, Ul-rich A, Standop J, Ding XZ, Adrian TE, Pour PM: Prevention of pancreatic cancer induc-tion in hamsters by metformin. Gastroenter-ology 2001; 120: 1263–1270.

99 Hawley SA, Boudeau J, Reid JL, Mustard KJ, Udd L, Makela TP, Alessi DR, Hardie DG: Complexes between the LKB1 tumor sup-pressor, STRAD alpha/beta and MO25 al-pha/beta are upstream kinases in the AMP-activated protein kinase cascade. J Biol 2003; 2: 28.

100 Apfeld J, O’Connor G, McDonagh T, Diste-fano PS, Curtis R: The AMP-activated pro-tein kinase AAK-2 links energy levels and insulin-like signals to lifespan in C. elegans . Genes Dev 2004; 18: 3004–3009.

101 Harkness TA, Shea KA, Legrand C, Brah-mania M, Davies GF: A functional analysis reveals dependence on the anaphase-pro-moting complex for prolonged life span in yeast. Genetics 2004; 168: 759–774.

102 Tschape JA, Hammerschmied C, Muhlig-Versen M, Athenstaedt K, Daum G, Kretzschmar D: The neurodegeneration mutant lochrig interferes with cholesterol homeostasis and Appl processing. EMBO J 2002; 21: 6367–6376.

103 Homburg R: Management of infertility and prevention of ovarian hyperstimulation in women with polycystic ovary syndrome. Best Pract Res Clin Obstet Gynaecol 2004; 18: 773–788.

104 Orchard TJ, Temprosa M, Goldberg R, Haffner S, Ratner R, Marcovina S, Fowler S: The effect of metformin and intensive life-style intervention on the metabolic syn-drome: the Diabetes Prevention Program randomized trial. Ann Intern Med 2005; 142: 611–619.

105 Barger JL, Walford RL, Weindruch R: The retardation of aging by caloric restriction: its significance in the transgenic era. Exp Gerontol 2003; 38: 1343–1351.

106 Miller RA, Buehner G, Chang Y, Harper JM, Sigler R, Smith-Wheelock M: Methio-nine-deficient diet extends mouse lifespan, slows immune and lens aging, alters glu-cose, T4, IGF-I and insulin levels, and in-creases hepatocyte MIF levels and stress re-sistance. Aging Cell 2005; 4: 119–125.

107 Richie JP, Jr., Leutzinger Y, Parthasarathy S, Malloy V, Orentreich N, Zimmerman JA: Methionine restriction increases blood glu-tathione and longevity in F344 rats. FASEB J 1994; 8: 1302–1307.

108 Orentreich N, Matias JR, DeFelice A, Zim-merman JA: Low methionine ingestion by rats extends life span. J Nutr 1993; 123: 269–274.

109 Ooka H, Segall PE, Timiras PS: Histology and survival in age-delayed low-trypto-phan-fed rats. Mech Ageing Dev 1988; 43: 79–98.

110 Segall PE, Timiras PS: Patho-physiologic findings after chronic tryptophan deficien-cy in rats: a model for delayed growth and aging. Mech Age Dev 1976; 5: 109–124.

111 De Marte ML, Enesco HE: Influence of low tryptophan diet on survival and organ growth in mice. Mech Age Dev 1986; 36: 161–171.

112 Goodrick CL, Ingram DK, Reynolds MA, Freeman JR, Cider NL: Effects of intermit-tent feeding upon growth, activity, and lifespan in rats allowed voluntary exercise. Exp Aging Res 1983; 9: 203–209.

113 Dhahbi JM, Mote PL, Wingo J, Rowley BC, Cao SX, Walford R, Spindler SR: Caloric re-striction alters the feeding response of key metabolic enzyme genes. Mech Age Dev 2001; 122: 35–50.

114 Dhahbi JM, Mote PL, Cao SX, Spindler SR: Hepatic gene expression profiling of strep-tozotocin-induced diabetes. Diabetes Tech-nol Ther 2003; 5: 411–420.

115 Bartke A: Long-lived Klotho mice: new in-sights into the roles of IGF-1 and insulin in aging. Trends Endocrinol Metab 2006; 17: 33–35.

116 Scharf JG, Braulke T: The role of the IGF axis in hepatocarcinogenesis. Horm Metab Res 2003; 35: 685–693.

117 Sedlaczek N, Hasilik A, Neuhaus P, Schup-pan D, Herbst H: Focal overexpression of insulin-like growth factor 2 by hepatocytes and cholangiocytes in viral liver cirrhosis. Br J Cancer 2003; 88: 733–739.

118 Yao X, Hu JF, Daniels M, Yien H, Lu H, Sharan H, Zhou X, Zeng Z, Li T, Yang Y, Hoffman AR: A novel orthotopic tumor model to study growth factors and onco-genes in hepatocarcinogenesis. Clin Cancer Res 2003; 9: 2719–2726.

119 Bartke A, Brown-Borg H: Life extension in the dwarf mouse. Curr Top Dev Biol 2004; 63: 189–225.

120 Dunn SE, Kari FW, French J, Leininger JR, Travlos G, Wilson R, Barrett JC: Dietary re-striction reduces insulin-like growth factor I levels, which modulates apoptosis, cell proliferation, and tumor progression in p53-deficient mice. Cancer Res 1997; 57: 4667–4672.

121 Hopfner M, Huether A, Sutter AP, Baradari V, Schuppan D, Scherubl H: Blockade of IGF-1 receptor tyrosine kinase has antineo-plastic effects in hepatocellular carcinoma cells. Biochem Pharmacol 2006; 71: 1435–1448.

122 Caro JF, Poulos J, Ittoop O, Pories WJ, Flickinger EG, Sinha MK: Insulin-like growth factor I binding in hepatocytes from human liver, human hepatoma, and normal, regenerating, and fetal rat liver. J Clin Invest 1988; 81: 976–981.

123 Baserga R, Peruzzi F, Reiss K: The IGF-1 re-ceptor in cancer biology. Int J Cancer 2003; 107: 873–877.

124 Cariani E, Lasserre C, Seurin D, Hamelin B, Kemeny F, Franco D, Czech MP, Ullrich A, Brechot C: Differential expression of in-sulin-like growth factor II mRNA in hu-man primary liver cancers, benign liver tu-mors, and liver cirrhosis. Cancer Res 1988; 48: 6844–6849.

Screening Potential Longevity Therapeutics

Gerontology 2007;53:306–321 321

125 Park BC, Huh MH, Seo JH: Differential ex-pression of transforming growth factor al-pha and insulin-like growth factor II in chronic active hepatitis B, cirrhosis and he-patocellular carcinoma. J Hepatol 1995; 22: 286–294.

126 Nardone G, Romano M, Calabro A, Pedone PV, de S, I, Persico M, Budillon G, Bruni CB, Riccio A, Zarrilli R: Activation of fetal promoters of insulin-like growth factors II gene in hepatitis C virus-related chronic hepatitis, cirrhosis, and hepatocellular car-cinoma. Hepatology 1996; 23: 1304–1312.

127 Scharf JG, Ramadori G, Dombrowski F: Analysis of the IGF axis in preneoplastic hepatic foci and hepatocellular neoplasms developing after low-number pancreatic is-let transplantation into the livers of strep-tozotocin diabetic rats. Lab Invest 2000; 80: 1399–1411.

128 Schirmacher P, Held WA, Yang D, Chisari FV, Rustum Y, Rogler CE: Reactivation of insulin-like growth factor II during hepa-tocarcinogenesis in transgenic mice sug-gests a role in malignant growth. Cancer Res 1992; 52: 2549–2556.

129 Scharf JG, Schmidt-Sandte W, Pahernik SA, Ramadori G, Braulke T, Hartmann H: Characterization of the insulin-like growth factor axis in a human hepatoma cell line (PLC). Carcinogenesis 1998; 19: 2121–2128.

130 Tsai TF, Yauk YK, Chou CK, Ting LP, Chang C, Hu CP, Han SH, Su TS: Evidence of autocrine regulation in human hepato-ma cell lines. Biochem Biophys Res Com-mun 1988; 153: 39–45.

131 Verspohl EJ, Maddux BA, Goldfine ID: In-sulin and insulin-like growth factor I regu-late the same biological functions in HEP-G2 cells via their own specific receptors. J Clin Endocrinol Metab 1988; 67: 169–174.

132 Tsai TF, Yauk YK, Chou CK, Ting LP, Chang C, Hu CP, Han SH, Su TS: Evidence of autocrine regulation in human hepato-ma cell lines. Biochem Biophys Res Com-mun 1988; 153: 39–45.

133 Verspohl EJ, Maddux BA, Goldfine ID: In-sulin and insulin-like growth factor I regu-late the same biological functions in HEP-G2 cells via their own specific receptors. J Clin Endocrinol Metab 1988; 67: 169–174.

134 Duclos MJ, Chevalier B, Upton Z, Simon J: Insulin-like growth factor-I effect on chick-en hepatoma cells (LMH) is inhibited by endogenous IGF-binding proteins. Growth Horm IGF Res 1998; 8: 97–103.

135 Zahradka P, Werner J, Yau L: Expression and regulation of the insulin-like growth factor-1 receptor by growing and quiescent H4IIE hepatoma. Biochim Biophys Acta 1998; 1375: 131–139.

136 Alexia C, Fallot G, Lasfer M, Schweizer-Groyer G, Groyer A: An evaluation of the role of insulin-like growth factors (IGF) and of type-I IGF receptor signalling in he-patocarcinogenesis and in the resistance of hepatocarcinoma cells against drug-in-duced apoptosis. Biochem Pharmacol 2004; 68: 1003–1015.

137 Su Q, Liu YF, Zhang JF, Zhang SX, Li DF, Yang JJ: Expression of insulin-like growth factor II in hepatitis B, cirrhosis and hepa-tocellular carcinoma: its relationship with hepatitis B virus antigen expression. Hepa-tology 1994; 20: 788–799.

138 Kim SO, Park JG, Lee YI: Increased expres-sion of the insulin-like growth factor I (IGF-I) receptor gene in hepatocellular car-cinoma cell lines: implications of IGF-I re-ceptor gene activation by hepatitis B virus X gene product. Cancer Res 1996; 56: 3831–3836.

139 Tao X, Shen D, Ren H, Zhang X, Zhang D, Ye J, Gu B: Hepatitis B virus X protein acti-vates expression of IGF-IR and VEGF in he-patocellular carcinoma cells. Zhonghua Gan Zang Bing Za Zhi 2000; 8: 161–163.

140 Scharf JG, Knittel T, Dombrowski F, Muller L, Saile B, Braulke T, Hartmann H, Rama-dori G: Characterization of the IGF axis components in isolated rat hepatic stellate cells. Hepatology 1998; 27: 1275–1284.

141 Biddinger SB, Kahn CR: From mice to men: insights into the insulin resistance syn-dromes. Annu Rev Physiol 2006; 68: 123–158.

142 Bilodeau M, Tousignant J, Ethier C, Roche-leau B, Raymond VA, Lapointe R: Anti-apoptotic effect of insulin on normal hepa-tocytes in vitro and in vivo. Apoptosis 2004; 9: 609–617.

143 Kosaki A, Webster NJ: Effect of dexameth-asone on the alternative splicing of the in-sulin receptor mRNA and insulin action in HepG2 hepatoma cells. J Biol Chem 1993; 268: 21990–21996.

144 Vella V, Pandini G, Sciacca L, Mineo R, Vi-gneri R, Pezzino V, Belfiore A: A novel au-tocrine loop involving IGF-II and the insu-lin receptor isoform-A stimulates growth of thyroid cancer. J Clin Endocrinol Metab 2002; 87: 245–254.

145 Sciacca L, Mineo R, Pandini G, Murabito A, Vigneri R, Belfiore A: In IGF-I receptor-de-ficient leiomyosarcoma cells autocrine IGF-II induces cell invasion and protection from apoptosis via the insulin receptor iso-form A. Oncogene 2002; 21: 8240–8250.

146 Kalli KR, Falowo OI, Bale LK, Zschunke MA, Roche PC, Conover CA: Functional insulin receptors on human epithelial ovar-ian carcinoma cells: implications for IGF-II mitogenic signaling. Endocrinology 2002; 143: 3259–3267.

147 Pandini G, Medico E, Conte E, Sciacca L, Vigneri R, Belfiore A: Differential gene ex-pression induced by insulin and insulin-like growth factor-II through the insulin receptor isoform A. J Biol Chem 2003; 278: 42178–42189.

148 Pandini G, Conte E, Medico E, Sciacca L, Vigneri R, Belfiore A: IGF-II binding to in-sulin receptor isoform A induces a partially different gene expression profile from insu-lin binding. Ann NY Acad Sci 2004; 1028: 450–456.

149 Frasca F, Pandini G, Scalia P, Sciacca L, Mineo R, Costantino A, Goldfine ID, Bel-fiore A, Vigneri R: Insulin receptor isoform A, a newly recognized, high-affinity insu-lin-like growth factor II receptor in fetal and cancer cells. Mol Cell Biol 1999; 19: 3278–3288.