The elderly population of the world increasing every year this increase in the elderly population is a cause of concern for health care systems around the world. One of the ways to combat this situation is nutritional intervention using specially designed food products for elderly people which would help improve the life quality of this class of people. It is well documented that various food products like berries which high nutritional value are known to have lot of beneficial effects. Cranberries and cranberry products have shown to have significant health benefits on cancer and heart disease prevention. In some recent studies it has been shown that cranberry can increase the life span of certain model organisms (Zou et.al).This branch of biology where in we study how nutrients cause genetic variations is known as Nutragenetics (phytochemicals).This study helps to gain insights in the processes by which these nutrients bring about changes in our health and this can help us to optimize the best possible combination of nutrients that can be used to improve our health and well being. Aging is influenced by various factors and one of the ways to combat is to take a balanced diet. However, it is difficult to assess the beneficial effects of these components on aging process in humans by using short-term dietary supplementation studies due to the high cost, duration and complications involved.
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Fig 1: Shows the various factors affecting the aging process and how beneficial nutrients help to reverse the aging process
To fully understand the efficacies of cranberry consumption on longevity, we propose to examine the effects of cranberry supplementation on major life span determinants, using the genetic model C .elegans which is a free living, soil dwelling nematode. C. elegans has become an attractive model organism for many reasons, which include its short lifespan, easy handling maintenance, and availability of full genome sequence data, ease of manipulation of its genome, homology with the human genome and the existence of a good collection of mutants.
C. elegans life cycle at 22ËšC (artwork by Altun and Hall, © Wormatlas)
The benefits of cranberries and cranberry products on longevity have emerged recently. However, little is known about the roles of cranberry on life span regulation mechanistically. It will be interesting to find out if cranberry can extended the lifespan in C elegans or not and does cranberry supplementation effects the normal behavior of the worms.
Numerous researches has shown that cranberries and cranberry products contain significant amounts of antioxidants and other phytonutrients that may help protect against human diseases, such as urinary tract infections, heart disease and cancer [1-3]. Recently, more evidence has emerged to demonstrate the health benefits of cranberry on aging and age-related diseases . However, little is known about the functions of cranberry on life span regulation, which are of great interest for customers and the public health agencies. Toward this goal, investigation of the effects of cranberry on major life span determinants is extremely important and straightforward. To date, studies in aging and longevity have found a complex array of genetic and environmental factors, determining the life span of organisms. Of these determinants, environmental stress, insulin signaling and Sir2 proteins play important roles in longevity and therefore, stand out [5-10].
All cells and organisms are constantly exposed to alterations in their environment. Some of these environmental changes (stresses) will lead to cell lesions by disrupting cellular and molecular activities. In fact, aging is the accumulation of diverse progressive deteriorations in the cells and tissues, which ultimately affect the life span of organisms [11, 12]. Therefore, longevity and environmental stress are causally linked. Notably, environmental stress per se is capable of not only causing cell lesions, but also triggering endogenous response mechanisms to repair stress-induced damage and decline stress. Remarkably, damage fixation and normal function restoration is not the end of stress and stress response, while the elevated stress adaptation and resistance will further benefit the longevity of organisms. Research in aging has provided evidence to claim that organisms which were challenged by certain stress exhibited an extended life span when the stress was removed [13, 14].
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Insulin/IGF signaling (IIS) pathway affects longevity in diverse species from yeast to mammal. In C. elegans, initiation of the signaling cascade occurs when DAF-2, the sole insulin/IGF-1 receptor, binds to insulin-like ligands. Activated DAF-2 recruits AGE-1, a phosphatidylinositol 3-kinase (PI(3)K) . This kinase is antagonized by DAF-18, a homolog of human PTEN with IP3 phosphatase activity . Subsequently, the PIP3 activates the AKT-family kinases  and SGK-1 in a PDK-1-kinase-dependent manner . These active kinases inactivate the transcription factor DAF-16 by phosphorylation and keep it out of the nucleus [7, 8, 20], rendering it incapable of promoting or repressing gene expression (Fig.1). Thus, multiple effector kinases of the DAF-2 signaling pathway converge to negatively regulate DAF-16 activity by changing its localization within the cell.
Model describing the role of Ins/IGF-1 signaling in lifespan extension
(Mol Genet Genomics (2007) 277:601-617)
Sir2 proteins are evolutionarily conserved proteins and function as protein deacetylase or ADP-ribosyl transferase to post-translationally modify many targets, which in turn regulate a broad constellation of biological processes through the epigenetic control of gene expression [21-25]. Interestingly, many of these processes have direct and indirect effects on aging. In addition, it is notable that Sir2 proteins can regulate lifespan and aging processes partly through modulating Caloric Restriction pathways, and linking the nutritional status of the cell to transcriptional regulation. This unique trait of Sir2 proteins in diet makes them ideal indicators of longevity and aging.
Cranberry contains high levels of polyphenolic compounds, which have been shown to have numerous therapeutic or preventive properties, including anti-inflammation, anti-carcinogenesis and anti-cardiovascular dysfunction, partly through their anti-oxidant potentials [3, 26, 27]. Moreover, it has been demonstrated that cranberry may have anti-diabetes and anti-hypertension properties, possibly through the inhibition of α-glucosidase and α-amylase . Cell culture experiments indicate that cranberry can antagonize the cell toxicity induced by Aβ . Recently, Dr. Zou's laboratory in NIA has investigated the anti-aging effects of cranberry containing fruit extract in Mexican fruit fly (Mexfly). They found that cranberry containing extract can significantly extend life span of Mexfly by 10-15% in a diet composition dependent manner. This suggests that cranberry consumption has prolongevity property. However, it remains to be determined whether the anti-aging effect of cranberry consumption is conserved in other organisms and what the underlying mechanisms are.
Our laboratory is conducting aging research by employing C. elegans, which is the primary model for genetic research on aging, and many significant results obtained in this model have been confirmed in other species. Since almost all of crucial life span determinants we have identified so far are conserved in C. elegans, we propose to investigate the functions of cranberry on longevity in C.elegans, to see whether cranberry modulates the life span of worms by interfering with these determinants. Interestingly, collaboration with Dr. Sige Zou's laboratory in NIA, we found that limited amount of cranberry (0.1% cranberry supplementation) is capable of significantly increasing life span of worms by 25-30%, while higher amount of cranberry supplementation (1%) will shorten life span of worms (Fig 2). These preliminary results are encouraging, and we believe that accomplishment of the specific aims in this proposal will dramatically expand our awareness of long-term health benefits and functions of cranberry in longevity. Thus, this research will not only provide the scientific information of cranberry in worms' aging, but also offer clinical guidance to human health and disease prevention in the future.
To investigate the potential role of cranberry on promoting longevity, thus to further evaluate its health benefits in human, we propose to study the effects of cranberry on life span regulation by employing C. elegans as a model. Toward this goal, we will address the following three specific aims:
Specific Aim 1: To systematically assess the effect of cranberry consumption on the longevity of C.elegans.
Specific Aim 2: To evaluate the effect of cranberry supplementation on C.elegans resistance to environmental stress. Environmental stresses have been recognized as important determinants of life span.
Specific Aim 3: To investigate the effect of cranberry on insulin/IGF signaling, which is evolutionarily conserved in animals to regulate aging and longevity, and SIR-2.1, another conserved key regulator of aging in animals.
Determination of Optimum concentration:
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The initial round of screening for finding out the optimum concentration of cranberry, which would show the maximum increase in the lifespan of C.elegans was found out by subjecting the worms to various different concentration of pure cranberry extract and then carrying out a lifespan assay.
It was found out that at higher concentration i.e. 2%, 1%.The worms died rapidly as compared to the worms maintained at lower concentrations of cranberry. This was thought to be due to cranberry's acidic nature which might be detrimental to the worms.
The Final concentration which was found out to show a significant increase in lifespan was 0.1% of cranberry extract. It was also found out that at higher concentration of cranberry the worms tend to lay more eggs, because in some previous studies it has been shown that the abolition of germline cells can increase the lifespan of worms significantly (Hsin and Kenyon 1999; Arantes-Oliveira et al. 2002, 2003) .This was thought to be one of the reasons for a decrease in lifespan of the worms at higher concentrations of cranberry. For finding out this a lifespan assay was carried out using fem-1 mutants which are germline defective worms, it was found out that these worms didn't show any significant difference in lifespan of the worms. Which means that the egg laying capacity is probably not correlated to lifespan of the worms in this particular case.
Fig: Lifespan curves of C elegans supplemented with cranberry carried out at 25 C. Supplementation of 0.1% of cranberry significantly increases the mean lifespan from 14.2 days to 17.9 days (p< 0.0001).Supplementation of 1% of cranberry significantly decreases the mean lifespan of C elegans from 14.2 days to 10 days (p<0.0001)
Fig: Shows the lifespan assay curves for wild type N2 and fem-1 worms when treated with different concentrations of cranberry at 25 C.
Data from initial rounds of stress assays:
The cranberry supplemented worms were subjected to different types of stress conditions namely Heat shock, oxidative stress and U.V. It was found out that there was a significant increase in the lifespan of the cranberry supplemented worms as compared to the worms which were not supplemented with a cranberry diet when the worms were subjected to heat shock treatment at 37 C for three and a half hours.
Although it was thought that cranberry supplementation would increase the lifespan of the worms when subjected to oxidative stress and U.V, a similar result was not observed i.e. either there was no change in the lifespan or it decreased the lifespan of the worms. Which led us to believe that cranberry supplementation with the stress treatment might have been producing by products which are probably toxic for the worms. Hence, in the next series of lifespan assay which will be carried out we will use cranberry treated wild type N2 worms (L4 stage) for the assay and the plates on which the stress assay will be carried out will not be supplemented with cranberry.
Fig the worms supplemented with cranberry at 37 C had a marked increase increase in lifespan as compared to the worms which were not treated with cranberry at 37 C
Pathways affecting the lifespan extension process due to cranberry supplementation:
To understand the mechanism as to how the lifespan extension was taking place when the worms were supplemented with cranberry we carried out a series of lifespan assay using various mutant worms, which are know be the key regulators in the aging process in C . elegans. We used the following worms strains for carrying out these assay VC199,GR1352
It was observed that although there is an increase in the increase of the lifespan of some of the worms treated with cranberry i.e. VC 199 and GR1352, the difference was not significant enough. Because, of this a few more rounds of the assay has to be carried out to determine which pathways play an important role to bring about this extension in lifespan of the worms.
Specific Aim 1: To systematically assess the effect of cranberry consumption on longevity of C.elegans.
Our preliminary data indicates that 0.1% cranberry extract prolongs the life of the wild type N2 worms, while 1% cranberry extract shortens their lives. To comprehensively evaluate the effect of cranberry on longevity in C.elegans, we supplemented a series of concentration of cranberry extract to N2 worms and monitored their life spans. Specifically, synchronized young adults were transferred to bacteria OP50 seeded NGM plates, which contain cranberry as supplementation up to 2%. In detail, the concentrations of 0.01%, 0.02%, 0.04%, 0.06%, 0.08%, 0.1%, 0.2%, 0.5%, 1%, 1.5% and 2% were tested, comparing to the control without cranberry. Subsequently, life span analysis was performed and the appropriate amount of cranberry consumption for prolongevity was explored in worms. Thus, this assessment provided an overview of the correlation between cranberry consumption and health benefits, especially in longevity.
Specific Aim 2: To evaluate the effect of cranberry supplementation on C.elegans resistance to environmental stresses and the mechanisms involved:
Environmental stresses have been recognized as important determinants of life span. Most of long lived animals show dramatic resistance to various environmental stresses, including thermal alteration, ultraviolet irradiation, oxidative stress, etc [30-34]. In this aim, we will comprehensively evaluate the effects of cranberry on C. elegans stress response. Briefly, we will maintain healthy wild type N2 worms for at least two generations by supplementing proper amounts of cranberry in the medium, which is determined in specific aim 1. Thereafter, these well maintained worms will be treated with the above mentioned stress responses for which the conditions are mentioned below:
Heat Shock : the treated worms and non treated ones are placed at 37 C for three and half hours and then a lifespan assay is carried out at 25 C
Oxidative Stress: the treated and non treated worms will be transferred to the plates containing know optimized concentrations of paraquat, which is a strong oxidizing agent and then a lifespan assay will be carried out.
U.V : the treated worms and non treated worms will be subjected to U.V for which the conditions have been optimized and then a lifespan assay will be carried out.
As a consequence, life span assay, brood size analysis and locomotion behavior assessment will be carried out. Once the positive effect of Cranberry on stress resistance is found out, a further investigation will be carried out to find the mechanisms and pathways that work in this stress resistance process. A screening for genes/pathways responsible for this stress mechanism will be carried out once we find out the major pathways that brings about the lifespan extension process which will be determined in specific aim 3.For this the worm candidates found out from specific aim 3 will be used . Stress will be subjected to these particular worms and then a lifespan assay would be carried out to access the effect of these major lifespan extending pathways on stress resistance.
It has been shown in previous experiments that under oxidative stress lipid peroxidation takes place (Gibson et al. 1976).To check the effect of cranberry supplementation on this process we will compare the levels of control and treated worm for 4-Hydroxynonenol (4-HNE)which is a product of lipid peroxidation.
Another study that would be done to check the mRNA levels of the genes induced due the stress response and how this is affected by cranberry supplementation, a Real time-PCR analysis will be performed for this to find out the expression levels of the genes which are responsible for coding the mRNA for Heat shock proteins(HSPs) which is thought to increase due to the aging process(Lund et al 2002).A real time PCR will also be carried out to find the level of Super oxide dismutase(SOD) expression in worms under this stress response when supplemented with cranberry and without cranberry . Undoubtedly, this evaluation will generate a portfolio of the benefits of cranberry consumption on stress resistance.
Recently a lot of studies have been carried out to check the effect of cranberry on diabetes. One of the experiments which will be carried is to check the lifespan of worms supplemented with and without cranberry when glucose is added to the medium. If we some interesting results are observed, then a further investigation will be carried out to find out the mechanism of this process.
Expected results and pitfalls:
We expect to observe that the worms treated with cranberry should live an extended amount of time as compared to the worms which are not treated and are subjected to the stress conditions. But it was not what we observed in the case of oxidative stress and U.V treatment for which might be a limitation.
As it is well established that stress and aging are correlated we might observe certain important pathways playing a major role in this life extension process which will be determined in Aim 3. The major limitation in this case would be that we might not find any correlation with the pathways which are of interest and it might be a separate/new pathways which might bring about this stress resistance phenomenon.
Although we might observe a decrease in the levels of 4-HNE, but a major limitation here is that as of now there has been no studies which show a link between aging and 4-HNE levels.
We expect to observe that the levels of HSPs in the control worms increase markedly as compared to worms which are treated with cranberry. In the same way the levels of SOD should increase in the cranberry treated worms when treated with cranberry.
Specific Aim 3: To investigate the effect of cranberry on insulin/IGF signaling and SIR-2.1, two conserved key regulators of life span in animals.
In this aim, we will determine whether cranberry consumption will affect the functions of insulin/IGF signaling pathway or SIR-2.1, thus, to modulate the life span in worms. To test insulin/IGF signaling, worm mutants of daf-2, age-1, pdk-1 and akt-1 will be maintained by supplementing optimal amount of cranberry extract, and their life span analyses will be performed individually. If the life span is synergized and greater than the sum of the effect of cranberry and each of the mutants, it indicates that cranberry consumption won't affect insulin/IGF signaling. If the life span exhibits the same life span just as worm mutants, it indicates that cranberry consumption depends on insulin/IGF signaling to extend the life span of worms. Meanwhile, we will also test the activity of DAF-16 when N2 worms are treated with cranberry extract, since insulin/IGF signaling modulates life span through DAF-16. Briefly, we will employ real-time PCR to quantify the expression of sod-3, a target gene of DAF-16, representing the alterations of DAF-16 activity. Taken together, we will assess whether cranberry consumption affects insulin/IGF signaling to prolong the worms' life.
Previous studies have shown that either over expression or activity increase of SIR-2.1 extends worms' life span. Therefore, to investigate whether the life span extension by cranberry consumption is mediated by SIR-2.1, we will test SIR-2.1 expression level as well as its enzymatic activity when optimal amount of cranberry extract is supplied to wild type N2 worms. To examine SIR-2.1 expression, real-time PCR will be conducted to quantitate the transcriptional changes of sir-2.1 in cranberry treated worms versus none treated worms. To test protein deacetylase activity of SIR-2.1, SIR-2.1 antibody will be used to make affinity chromatography column to purify SIR-2.1 proteins from cranberry treated worms and none treated worms, respectively. Thereafter, the HDAC fluorescent activity assay/drug discovery kit (AK-500, BIOMOL Research Laboratories) will be employed to assay the deacetylase activity of SIR-2.1 .
One of the hallmarks of aging related cellular damage is the intracellular accumulation of Lipofuscin, which is an autofluorescent material that tends to accumulate in an aging cell. A study will be carried out in which cranberry treated worms which are 12 day old will be compared with non treated worms to check the levels of intestinal Lipofuscin.
Future line of studies:
For checking the cranberry supplementation plays any role in apoptosis due to stress a Tunnel assay can be carried out. For this the worms supplemented with cranberry and without cranberry, will be subjected to the stress conditions and the profile generated will be studied to check the effect of cranberry on DNA damage and apoptosis process.
As it is known that Mitochondrion plays a very important role in the aging process, it might be really interesting to study how cranberry supplementation effects the functioning of mitochondria. For doing this a Seahorse XF24 analyzer can be used which measures the rate of mitochondrial respiration.
In the future similar studies can be carried out on other food products which are known to have potential benefits and a portfolio can be generated which will help us to gain a clear understanding about the whole process. This will help to us to eat better and live healthier.