Alobar Greywalker (alobar) wrote in longevity,
Alobar Greywalker
alobar
longevity

Reaching for Near Immortality with my Critter Companions!

        I came upon a nice long article which talks about life extension of fruit flies using Rhodiola.  I am assuming similar life extension properties of Rhodiola would benefit humans.

       Website is long and filled with technical detail which do not interest me. Myriad graphs as well. So go to the website if interested.

=====================================

Extension of Drosophila Lifespan by Rhodiola rosea
through a Mechanism Independent from Dietary Restriction


Samuel E. Schriner, Kevin Lee, Stephanie Truong, Kathyrn T. Salvadora, Steven Maler, Alexander Nam,
Thomas Lee, Mahtab Jafari


Published: May 21, 2013
DOI: 10.1371/journal.pone.0063886

Abstract

Rhodiola rosea has been extensively used to improve physical and mental performance and to protect against stress. We, and others, have reported that R. rosea can extend lifespan in flies, worms, and yeast. However, its molecular mechanism is currently unknown. Here, we tested whether R. rosea might act through a pathway related to dietary restriction (DR) that can extend lifespan in a range of model organisms. While the mechanism of DR itself is also unknown, three molecular pathways have been associated with it: the silent information regulator 2 (SIR2) proteins, insulin and insulin-like growth factor signaling (IIS), and the target of rapamycin (TOR). In flies, DR is implemented through a reduction in dietary yeast content. We found that R. rosea extract extended lifespan in both sexes independent of the yeast content in the diet. We also found that the extract extended lifespan when the SIR2, IIS, or TOR pathways were genetically perturbed. Upon examination of water and fat content, we found that R. rosea decreased water content and elevated fat content in both sexes, but did not sensitize flies to desiccation or protect them against starvation. There were some sex-specific differences in response to R. rosea. In female flies, the expression levels of glycolytic genes and dSir2 were down-regulated, and NADH levels were decreased. In males however, R. rosea provided no protection against heat stress and had no effect on the major heat shock protein HSP70 and actually down-regulated the mitochondrial HSP22. Our findings largely rule out an elevated general resistance to stress and DR-related pathways as mechanistic candidates. The latter conclusion is especially relevant given the limited potential for DR to improve human health and lifespan, and presents R. rosea as a potential viable candidate to treat aging and age-related diseases in humans.

Introduction

The root extract of Rhodiola rosea, also known as the golden root, has been widely used in traditional and integrative medical practices in Europe and Asia, where it has been purported to mediate a variety of beneficial effects in humans, such as improved mood, improved physical and mental stamina, and enhanced protection against high altitude sickness [1]. The extract has also been reported to protect against tumor progression in mice, improve endurance in rats, improve blood glucose profiles in diabetic mice, and protect snail eggs against oxidative stress, heat, and heavy metals [2]–[5]. Our group has previously reported that R. rosea can extend the lifespan of the fruit fly, Drosophila melanogaster, protect flies and human cultured cells against oxidative stress, and decrease the production of reactive oxygen species in isolated fly mitochondria [6]–[8]. In addition to the fly, the extract has also been shown to extend lifespan in the worm, Caenorhabditis elegans [9], and in the yeast, Saccharomyces cerevisiae [10]. These observations demonstrate that R. rosea lifespan-extending properties are not limited to the fly, and suggest that it may be a viable treatment to slow aging and abrogate age-related diseases in a range of species, potentially including humans.

The molecular action of R. rosea is not known, though its effects in worms suggest that it may act through hormesis [9], where pretreatment of a mildly toxic compound induces defense systems that further protect the organism against any additional stress [11]. Contrary to this, we have shown that R. rosea is able to confer protection against oxidative stress in cultured cells at doses far below what is required to activate antioxidant defenses [8]. We then proposed an alternate hypothesis, that R. rosea may act through a pathway related to dietary restriction (DR), e.g., as a DR mimetic. To date, DR, defined as a decreased total caloric intake in the absence of malnutrition, is considered the most robust non-genetic treatment for improving health and extending lifespan in model organisms. This treatment has been shown to benefit nearly all organisms tested, from yeast to primates [12]–[15], though a recent study has questioned its effectiveness in primates [16]. Like R. rosea, the molecular mechanism of DR is not known, however, three different but overlapping molecular pathways, sometimes termed nutrient-sensing pathways, are thought to be involved in the mechanism of DR. These molecular pathways are the silent information regular 2 (SIR2) proteins [17], the target of rapamycin (TOR) [18], and insulin and insulin-like growth factor signaling [19]. Given the robustness of DR in model systems, there has been a significant effort to identify compounds that could mimic the action of DR at the molecular level. Three prominent suggested candidates are resveratrol, rapamyacin, and metformin [20]. Of these three, metformin, a highly prescribed medication for type 2 diabetes, appears to be the most promising. While metformin appears to mimic the molecular effects of DR in mice [21], it has had mixed results in invertebrate models; it extended mean lifespan in worms [22], but had no positive effect in flies [23]. Nevertheless, a plausible mode of action of R. rosea could be that it acts as a DR mimetic.

The purpose of this work was to examine such a possibility. We found that R. rosea extended lifespan independent of dietary yeast content in flies, the method by which DR is imposed in flies. The extract also extended lifespan when any of the 3 nutrient-sensing pathways were perturbed, demonstrating that it acts independently from these pathways as well. Rhodiola rosea exhibited no effect in male flies in 4 DR-related parameters examined: glycolysis, dSir2 expression, NAD+/NADH ratios, and total soluble protein levels. Though, in females the extract down-regulated glycolytic enzymes and elevated NAD+/NADH ratio, suggesting the possibility of a partial DR effect in females. Nonetheless, R. rosea was able to extend lifespan in flies while exhibiting no experimental outcome consistent with DR, refuting our original hypothesis, and demonstrating that R. rosea acts through a mechanism unrelated to DR.

Discussion

Previously, we had reported that the root extract of Rhodiola rosea could extend the lifespan of the fruit fly, Drosophila melanogaster [6], [7]. Our findings are supported by similar results in the worm, Caenorhabditis elegans [9], and in the yeast, Saccharomyces cerevisiae [10]. However, the molecular mechanism by which R. rosea extends lifespan is not known. Here, we examined whether R. rosea acts through molecular pathways associated with dietary restriction (DR), a reduction in caloric intake without malnutrition, which is considered to be the most robust mechanism for extending lifespan and improving health in model organisms [12]–[15]. Our results show that R. rosea acts in a manner unrelated to DR.

Dietary restriction is imposed in mammals by decreasing the amount of food provided [35]. However, in flies it is typically undertaken by decreasing the percentage of yeast in the diet [24]. Decreasing the dietary yeast content then increases lifespan up to a point where a further reduction begins to shorten lifespan (Figure 1). This latter shortening of lifespan is likely due to an excessive restriction of protein and/or sub-optimal intake of other nutrients. If a compound or extract acts in a DR-dependent manner, we would expect a maximal effect on lifespan at the highest dietary yeast content, and then a diminished effect as decreased dietary yeast concentrations increased lifespan (Figure 1, controls). We would also expect that a DR effect would further compromise lifespan at the lowest dietary yeast content, as the animals are in a nutritionally deprived state. Such a DR-dependent mode of action has been clearly demonstrated for the target of rapamycin (TOR) [18]. However, in our work, R. rosea extended lifespan at all dietary yeast contents, even when low dietary yeast content shortens lifespan (Figure 1). This is contrary to our predictions of a DR-related effect. Furthermore, while the physiological effects of DR have been extensively documented, its molecular mechanism, like R. rosea, is not known. In addition to TOR mentioned above, two other molecular pathways have been implicated in action of DR: the silent information regulator 2 proteins (SIR2), and insulin and insulin-like growth factor signaling (IIS) [19], [26]. The ability of R. rosea to still extend lifespan in flies in which these pathways were perturbed (SIR2 and IIS were blocked, while TOR signaling was both blocked and activated) strongly supports a DR-independent mode of action (Figures 3 and 4).

To further explore a potential DR effect by R. rosea, we examined four other parameters associated with DR: down-regulation of glycolysis, up-regulation of dSir2, elevated NAD+/NADH ratios, and decreased soluble protein levels [25], [28]–[30], [34]. In male flies, R. rosea had no effect on any of these parameters (Figures 4A, C, and D, 5A, B, C, and G). Thus, in males, the examination of 8 different parameters (these 4 parameters plus dietary yeast content, and the 3 nutrient sensing pathways) showed no R. rosea induced DR effect. In females, we did see some DR-related effects: down-regulation of glycolytic genes (Figure 4B and C), and elevated NAD+/NADH ratios (Figure 5C). However R. rosea did not decrease soluble protein levels in females (Figure 5G), and actually decreased dSir2 expression (Figure 4D), both effects inconsistent with DR. Despite the mixed results in females, the ability of R. rosea to extend lifespan in males without any associated DR effects, demonstrates that R. rosea is fully capable of extending lifespan in a DR-independent manner.

The feeding of R. rosea to flies did lead to some surprising results such as the decrease in water content (Figure 5I). Dehydration is thought to be a significant contributor to fly death, and selection for postponed aging has been found to result in an increased tolerance to desiccation [36]. Since R. rosea increases lifespan, we expected that if it had any effect on water content it would be an increase. This decreased water content may have resulted from displacement of water by the elevated fat seen in R. rosea-fed flies (Figure 5H). The altered water and fat contents, the lack of any effect on desiccation and starvation tolerance, and the inability to protect against heat were surprising observations given the evidence that R. rosea protects against stress in a variety of conditions [4], [5], [7], [8]. These types of results are not unique to R. rosea. For instance, the finding that mitochondrial HSP22 was down-regulated is similar to what we observed with another botanical, Rosa damascena, which also extended fly lifespan [37]. While we have previously shown that R. rosea protected against oxidative stress in flies and cultured cells [7], [8], the role of oxidative stress in aging has been somewhat marginalized recently [38], [39]. In flies, it has been argued that elevated oxidative stress resistance is valuable only in shorter-lived strains, and loses its beneficial effects in longer-lived, presumably healthier flies [40]. Given our results that R. rosea extends lifespan in very long-lived DR-treated flies (Figures 1 and 2), we could argue that even the protection against oxidative stress imparted by the extract may be unimportant in its ability to extend lifespan.

An interesting and important observation is the down-regulation of dilp2, 3, and 5 (Figure 5D, E, and F). We argue that R. rosea does not act through IIS, due to its ability to extend lifespan when the pathway is blocked by the absence of the insulin receptor substrate, chico (Figure 3A and B). Nevertheless, the down-regulation of these proteins may be instrumental in its action, as their expression levels, particularity those of dilp2, are inversely related to lifespan in fruit flies. For example, over-expression of dilp6 in the fat body extended fly lifespan and decreased the expression levels of both dilp2 and 5 [41], while over-expression of dFOXO, which also extends lifespan, decreased the expression levels of dilp2 [31]. Over-expression of uncoupling protein-3 elevated DILP2 protein levels and shortened lifespan [32]. The most direct test of the role of these proteins in aging was the deletion of the neurosecretory cells that produce dilp2, 3, and 5, which resulted in an extended lifespan [42]. However, the phenotypes observed in this experiment only partially overlap those seen in flies fed R. rosea. Flies that lacked dilp2, 3, and 5 expression had an elevated fat content, were sensitized to heat, and exhibited enhanced protection against oxidative stress and starvation [42]. In flies fed R. rosea, we did see protection against oxidative stress [7] and an elevated fat content (Figure 5H), but we saw no protection against starvation and no sensitivity to heat (Figure 6E and F). Actually, in our case, females had an enhanced tolerance to heat (Figure 6F. Therefore, it isn’t clear exactly what the role of decreased dilp expression is in the action of R. rosea. A possible explanation for the differences in outcomes between Broughton’s experiment and ours is the degree in which the dilp2, 3, and 5 are down-regulated. In their case, the expression of these genes are completely absent, while in ours their expression levels are decreased to 25–50% of baseline. Alternatively, R. rosea may act on other targets, which might mitigate the effects of decreased dilp expression. Future experiments in flies with elevated or decreased dilp expression levels will help inform us on the role of the dilp2, 3, and 5 in the action of R. rosea.

The root extract of R. rosea used in this study is composed of at least 140 compounds [43]. The active compounds in the extract are not known for certain, though they are thought to include salidroside, tyrosol and 3 rosavin compounds [1]. The extract also has a significant polyphenol content that may contribute to its activity [43]. While we are currently searching for the extract’s active compound with respect to lifespan extension, it may be that there is no single compound responsible, and such a search may be somewhat misguided due to the complexity of aging. There are likely thousands of genes that play role in the lifespan of an organism [44]. Therefore, it may not be particularly successful to search for single anti-aging compounds, as most compounds would have a limited number of molecular targets. One exception would be compounds that mimic DR, as such molecules could modulate the expression of many downstream targets. However, the recent finding that DR may not extend lifespan in primates [16] may lower the enthusiasm regarding DR mimetic compounds. The use of complex natural products to screen for lifespan-extending effects then presents an advantage, as these extracts contain many molecules that could simultaneously target multiple molecular pathways.

Our initial hypothesis was that R. rosea extended fly lifespan by acting as a DR mimetic by targeting one of 3 DR-related pathways. Here, we tested this hypothesis through a series of genetic, dietary, and phenotypic analyses. As a result of our findings, we argue that R. rosea extends fly lifespan through a mechanism independent from DR, though that mechanism has not yet been identified. We also show that R. rosea extended lifespan in both sexes. Many aging studies in Drosophila are conducted only with males, and often when both sexes are used, sex-specific differences are observed. For example, the complete loss of chico extended lifespan only in females [45], whereas Rosa damascena and curcumin both showed sex-specific effects on lifespan depending on the strain used [37], [46]. The ability of R. rosea to extend lifespan in DR-treated flies shows that the extract acts in long-lived, presumably very healthy flies, and doesn’t simply make sick flies healthier. Of particular note is our finding that R. rosea feeding when combined with a low dietary yeast content (0.3%) resulted in some of the longest lived laboratory flies, with mean lifespans of over 90 days and maximum lifespans exceeding 120 days in both sexes (Figure 1 and Table S1). Thus, the extract has the potential to improve longevity in humans of both genders who are healthy and non-obese. The additive activity of DR and R. rosea also shows that the extract could be used in conjunction with known DR-mimetics, or other compounds which target DR-related pathways, to provide even further benefits in obese or sick individuals. Therefore, R. rosea may present a viable and potentially powerful therapy for aging and age-related diseases in a broad group of patients. Our future work will be to identify the precise molecular target of the extract using other genetic models in combination with microarray and proteomic techniques, identify its active compounds, and study its interaction with other known lifespan extending treatments.

Feeding and Lifespan Assays

Flies were fed R. rosea extract based on the methods described in Jafari et al. 2007 [6]. Concentrations of R. rosea extract described were dissolved in a yeast solution (4% yeast in 1% acetic acid), and 75 µL of this mixture was overlaid on a banana-molasses food composed of 9% carbohydrate content and a 3.6% yeast content. For the DR studies, dietary yeast contents, in both the banana-molasses food and in the overlaid yeast solution, were as indicated in Figure 1, the concentrations of all other dietary components were unchanged. Flies were maintained at 22±1°C under a 12 h light: 12 h dark cycle for all experiments. For lifespan studies, flies were housed 12 per vial (6 males and 6 females). This density was maintained as long as feasible. Flies were given fresh food every two days and deaths were recorded at these times. Flies were fed R. rosea extract at a dose of 25 mg/mL, what we consider the optimal as it confers a maximal lifespan extension in both sexes [7]. Survival analyses were calculated based on the number of deaths recorded and evaluated by the log-rank Mantel-Cox test. Flies were also housed 12 per vial (6 males and 6 females) for all other experiments, independent of the total number needed, and transferred to fresh food every other day.
  • Post a new comment

    Error

    default userpic

    Your IP address will be recorded 

  • 0 comments