Cacao Procyanidins-Induced Lifespan Extension in Caenorhabditis elegans in a Nervous System and CaMKII-Dependent Manner

Abstract

Procyanidins are gaining attention due to their potential health benefits. We found that cacao liquor procyanidin (CLPr) from Theobroma cacao seeds increased the lifespan of Caenorhabditis elegans, a representative model organism for aging studies. The genetic dependence of the lifespan-extending effect of CLPr was consistent with that of blueberry procyanidin, which is dependent on unc-43, osr-1, sek-1, and mev-1, but not on daf-16, sir-2.1, or skn-1. The lifespan-extending effect of CLPr was inhibited by neuron-specific RNA interference (RNAi) targeting unc-43 and pmk-1, and in worms with loss-of-function mutations in the odr-3, odr-1, or tax-4 genes, which are essential in sensory neurons, including AWC neurons. It was also inhibited in worms in which AWC neurons or AIB interneurons had been eliminated, and in worms with loss-of-function mutations in eat-4 or glr-1, which are responsible for glutamatergic synaptic transmission. These results suggest that the lifespan-extending effect of CLPr is dependent on the nervous system. In addition, it also requires unc-43 and pmk-1 expression in nonneuronal cells, as demonstrated by the experiments with RNAi in wild-type worms, the neuronal cells of which are not affected by systemic RNAi. The osr-1 gene is expressed in hypodermal and intestinal cells and regulates the response to osmotic stress along with unc-43/calcium/calmodulin-dependent protein kinase II and the p38 mitogen-activated protein kinase pathway. Consistent with this, CLPr improved osmotic stress tolerance in an unc-43- and pmk-1-dependent manner, and it was also dependent on AWC neurons. The lifespan-extending and osmotic-tolerance-improving activities were attributed to procyanidins with a tetrameric or higher-order oligomeric structure.

Procyanidins derived from plants are oligomeric polyphenolic compounds formed from flavan-3-ols, namely (+)-catechin and (−)-epicatechin. Procyanidins in edible plants such as cacao and apples have attracted much attention due to their potential health benefits (1). For example, daily consumption of dark chocolate may lower the risk of diabetes, hyperglycemia, and cardiovascular disease (2–4). Recent studies have reported that cacao extracts rich in flavanols can improve mood states in healthy middle-aged women (5).

Cacao liquor procyanidin (CLPr) prepared from the seeds of Theobroma cacao is used as an ingredient in chocolate and cocoa. It is rich in polyphenols, such as catechin and epicatechin, and procyanidins, such as procyanidin B2 (epicatechin-(4β-8)-epicatechin), procyanidin C1 (epicatechin-(4β-8)-epicatechin-(4β-8)-epicatechin), and cinnamtannin A2 (CTA2; epicatechin-(4β-8)-epicatechin-(4β-8)-epicatechin-(4β-8)-epicatechin) (6).

Cacao liquor procyanidin has been shown to exert potential health benefits, some of which derive from its antioxidative activity. For example, CLPr supplementation reduced oxidative stress and inhibited diabetes-induced cataract formation in model animals (7). On the other hand, there are some health benefits of CLPr that are unrelated to antioxidative activity. For example, dietary supplementation with CLPr suppressed hyperglycemia and obesity in mice fed a high-fat diet through the activation of AMP-activated protein kinase α (AMPKα), which promotes glucose transporter type 4 (GLUT4) translocation and up-regulates uncoupling protein expression in skeletal muscle and adipose tissue (8,9).

Although procyanidins with a polymeric structure larger than dimers have poor bioavailability (10), bioactivities have recently been reported for the tetrameric procyanidin CTA2 that has a molecular weight of 1 155 g/mol. CTA2 significantly ameliorates postprandial hyperglycemia in mice by promoting GLUT4 translocation to the plasma membrane through the activation of insulin- and AMPK-signaling pathways (11).

It has also been suggested that CTA2 may positively affect the nervous system. In rats, low doses of procyanidins activate promotive adrenaline receptors β₁, β₂, and α₁ through the sympathetic nervous system and increase blood flow (12). When given to adult mice in the short term, it may improve spatial memory by increasing neurogenesis in the dentate gyrus (13). Additionally, the administration of CTA2 to mice with disuse atrophy can activate sympathetic nerves, promote hypertrophy, and inhibit the progression of disuse muscle atrophy (14).

Although procyanidins have multiple beneficial effects, the underlying molecular mechanisms of most of the effects remain unclear.

Caenorhabditis elegans is a species of nematode that is approximately 1 mm in length and has 2 sexes: hermaphrodite and male. Under normal conditions, most individuals are hermaphrodites, and there are only a few males. Hermaphrodites have a limited number of sperm and can fertilize their own oocytes. C elegans is widely used as an animal model in biogerontological research (15). Forward and reverse genetic analyses in C elegans have revealed around a hundred genes and several physiological processes that modulate lifespan. These genes and processes play a role in hormonal signaling, including insulin/insulin-like growth factor 1 signaling, nutrient sensing, and other forms of signaling, such as the dietary restriction pathway and germline signaling (16–19).

In the present study, we investigated the biological activities of CLPr in C elegans and found that dietary supplementation of CLPr extended the lifespan of the worms in a nervous system-dependent manner. The hermaphrodites have 959 somatic nuclei, 302 of which are neuronal cells, including 12 pairs of amphid sensory neurons with ciliated dendrites (20). Among these, the AWC neurons are a pair of odor-sensing neurons consisting of asymmetrically differentiated cells, called AWC^ON and AWC^OFF, that exhibit different gene expression patterns (21). For the AWC^OFF identity, it is crucial to activate the pathway that involves calcium/calmodulin-dependent protein kinase II (CaMKII) and factors of the p38 mitogen-activated protein kinase (MAPK) cascade. Loss-of-function (lf) mutations of the genes unc-43/CaMKII, nsy-1/MAPKKK, and sek-1/MAPKK in this pathway result in the 2-AWC^ON/no-AWC^OFF phenotype. In addition to its role in differentiating neurons, unc-43/CaMKII also plays a role in the postembryonic stage (22).

Thus far, many polyphenols, including procyanidins, have been reported to increase the lifespan of C elegans (23–26). The stress resistance and longevity effects of polyphenols are conserved across species, as has been shown in yeasts, fruit flies, and mice (26). However, the genetic requirements for polyphenols to exert their lifespan-extending effects sometimes differ, and the lifespan-extension mechanisms may thus vary. For example, some mechanisms are dependent on daf-16/FOXO, while others are not (26). In C elegans, olfactory and gustatory neurons play a role in lifespan regulation (27), but the effects of polyphenols on these neurons remain unclear.

In the present study, we found that dietary CLPr supplementation extended the lifespan of C elegans in a manner that was dependent on unc-43/CaMKII and the nervous system. This lifespan-extending effect is due to procyanidins with a tetrameric or higher-order oligomeric structure.

Method

C elegans Strains and Bacterial Strains

The C elegans strains used in this study were listed in Supplementary Data. The Escherichia coli strains OP50 and HT115(DE3) were obtained from the Caenorhabditis Genetics Center (CGC). Worms were maintained at 20°C on a nematode growth medium (NGM) seeded with E coli OP50 according to standard culturing methods (28). Heat-killed bacteria were prepared by heating at 65°C for 30 minutes.

Procyanidins

Cacao liquor procyanidin was prepared from cacao liquor as previously described (6). The fractions with high and low degrees of polymerization (DP) were obtained by fractionating CLPr based on molecular size according to reference (29). The HPLC chromatograms and polyphenol compositions of CLPr, the low-DP, and high-DP fractions are shown in Supplementary Figure 1. CTA2 was purchased from Merck (Darmstadt, Germany). To perform the longevity assay, CLPr, the high- and low-DP fractions, and CTA2 were dissolved in DMSO at a concentration of 60 mg/mL.

Longevity Assay

The measurement of lifespan was typically carried out at 25°C or 20°C. Eggs were collected from adult hermaphrodites using alkaline sodium hypochlorite. They were allowed to hatch in an S-basal buffer at 20°C. The S-basal buffer consisted of 100 mM NaCl and 50 mM potassium phosphate (pH 6.0). After hatching, the synchronized L1 larvae were transferred to NGM agar plates that had been spread with E coli OP50. The plates were kept at the specified temperature until the larvae grew into adults. The longevity assays were typically performed multiple times as follows. On the first day of adulthood, 15 individuals were placed on each of more than 5 NGM agar plates containing either 10 µg/mL CLPr and 0.017% dimethyl sulfoxide (DMSO), or 0.017% DMSO alone as vehicle control. This concentration of CLPr was sufficient to extend lifespan, and up to 0.3% DMSO as a co-solvent had no effect. When using 5-fluorodeoxyuridine (FUdR), the final concentration was adjusted to 40 µM. The worms were transferred to new plates corresponding to each condition every other day, and the live and dead worms were counted. Worms were considered dead if they did not move or respond to gentle prodding with a platinum wire. Individuals were excluded from the sample if they climbed the plate wall, crawled into the agar, had fertilized eggs hatch inside their bodies, or had protruding gonads. Survival curves were created using the Kaplan–Meier method based on lifespan assay data both with and without CLPr, and the differences in survival rates were analyzed using OASIS 2 (30). The log-rank test was utilized to calculate p values. Survival curves with a p value of less than .05 compared to the vehicle control were considered significantly different.

RNA Interference

RNAi was performed using the feeding method (31). The E coli HT115(DE3) strains transformed with an expression plasmid for the double-stranded RNA (dsRNA) of unc-43, pmk-1, or odr-3 were obtained from Open Biosystems/Thermo Scientific (Waltham, MA, USA). The bacteria were cultured in a liquid LB medium containing 50 µg/mL of ampicillin for 8–18 hours. After that, they were spread on NGM plates that contained 1 mM isopropyl-β-d-thiogalactopyranoside and 50 µg/mL of ampicillin. These seeded plates were left at room temperature overnight to induce dsRNA expression and used for lifespan analysis.

Osmotic Stress Tolerance Assay

Synchronized L1 larvae were cultured at 25°C. For acute assays, on the first day of adulthood, the worms were transferred to fresh plates containing either 10 µM CLPr and 0.017% DMSO or just 0.017% DMSO alone. After 24 hours, the worms were collected and washed with an S-basal medium containing either 10 µM CLPr and 0.017% DMSO, or just 0.017% DMSO. After that, 50–100 worms were placed on NGM agar plates containing 500 mM NaCl. Statistical significance was evaluated by the log-rank test. The time to immobility was measured for each worm. For the chronic assay, the worms were transferred to fresh NGM plates upon reaching adulthood and cultured for 24 hours. After that, they were transferred to new NGM agar plates containing 500 mM NaCl and either 10 µM CLPr with 0.017% DMSO or just 0.017% DMSO. The plates were kept at 25°C, and the viability of the worms was evaluated after 72 hours. Survival rates were calculated as the average of 3 to 5 plates. Statistical significance was evaluated using the Student’s t test.

Measurement of gpdh-1_p::GFP Expression

Young adult VP198 worms carrying the gpdh-1_p::GFP transgene were cultured with or without CLPr for 24 hours. The GFP fluorescence images of the worms were then photographed using a DP52 digital CCD camera under an OLYMPUS SZX16 fluorescence microscope. Fluorescence intensity was measured using ImageJ software (National Institutes of Health). Statistical significance was evaluated by the Student’s t test.

Results

Dietary Supplementation With CLPr Extends the Lifespan of C elegans

To explore the beneficial activities of CLPr, we examined the effects of its dietary supplementation on the lifespan of C elegans. Worms fed heat-killed E coli lived longer than those fed live E coli. When given 10 µg/mL of CLPr, the mean adult lifespan was increased by 13% at 20°C regardless of whether they were fed live or killed bacteria (Figure 1A, Supplementary Table 1). In the subsequent experiments, the worms were given live bacteria as their food source.

Although a reduced caloric intake has been shown to extend the lifespan in various organisms (16), the lifespan-extending effect of CLPr did not appear to be due to a reduced caloric intake because CLPr supplementation did not affect the growth rate, body length, egg-laying rate, or total brood size (Supplementary Figure 2A and B). Additionally, it did not decrease pharyngeal pumping during the adult stage (Supplementary Figure 2C). Moreover, supplementation with CLPr resulted in a longer lifespan even in the eat-2(ad465) mutant, which is often used as a genetic model for studying dietary restriction (18) (Supplementary Figure 2D, Supplementary Table 2).

The lifespan of C elegans is known to be shorter at higher temperatures. Supplementation with CLPr increased the lifespan by 18% and 13% at 25°C and 20°C, respectively, but it did not significantly affect the lifespan at 15°C (Figure 1B and C, Supplementary Table 1). Therefore, the lifespan comparisons in this study were based on data obtained at 25°C or 20°C. In longevity assays, FUdR is often used to prevent the reproduction of C elegans. CLPr supplementation extended the lifespan regardless of the presence of FUdR.

CLPr-Induced Lifespan Extension Exhibits Genetic Requirements Consistent With the Procyanidin-Rich Fraction From Blueberry

To elucidate the mechanism underlying the CLPr-induced lifespan extension, the genetic requirements were investigated using various mutant strains carrying lf mutations (Figure 2A, Supplementary Figures 3A and 4, Supplementary Table 2). The results obtained for CLPr were compared to published results for various polyphenols (26), and they were found to be essentially the same as those reported for the procyanidin-enriched fraction from blueberry (BPr) (24). BPr at a concentration of 67 µg/mL increased the mean lifespan of C elegans by 16% at 20°C, and by 20% at 25°C with or without FUdR. This lifespan-extending effect was seen in the genetic backgrounds of sir-2.1(ok434), daf-16(mgDf50), and skn-1(zu67), but not in those of osr-1(rm1), unc-43(n498n1186), sek-1(ag1), or mev-1(kn1) at 25°C. These genetic dependencies were consistent with those observed for CLPr. However, sek-1(ag1) was only examined at 20°C because frequent internal hatching occurred at 25°C (32). In addition to sek-1(ag1), nsy-1(ks397), a lf mutant of nsy-1/MAPKKK that regulates sek-1/MAPKK in the p38 MAPK pathway, did not show lifespan extension when supplemented with CLPr. Therefore, it was concluded that, similar to BPr, CLPr extends the lifespan of C elegans in a manner that is dependent on osr-1, unc-43, and factors of the p38 MAPK pathway. This lifespan-extending effect was suppressed in the lf mutant pmk-1(kn25), suggesting a dependence on pmk-1/p38 MAPK.

CLPr-Induced Lifespan Extension Requires unc-43/CaMKII Expression in Nonneuronal and Neuronal Cells

Among the genes involved in the lifespan-extending effect of CLPr, we first focused on unc-43/CaMKII, which functions both during and after the embryonic period. At the embryonic stage, a signaling pathway involving unc-43 specifies the asymmetric identities of AWC neurons as AWC^ON and AWC^OFF (21,33,34). To determine whether postembryonic expression of unc-43 is necessary for CLPr-induced lifespan extension, RNAi knockdown was conducted using the feeding method (31). The E coli HT115(DE3) strain was used to produce dsRNA and was provided as a food source to the worms. When worms were fed bacteria that did not express dsRNA targeting specific mRNA, supplementation with 20 μg/mL of CLPr extended their lifespan by approximately 9% (Figure 2B, Supplementary Figure 5, Supplementary Table 3). This life-span extension was suppressed when the worms were fed bacteria expressing dsRNA targeting unc-43. Furthermore, it was also suppressed by the knockdown of pmk-1. As systemic RNAi is not effective in C elegans neurons (35), these results suggest that the expression of unc-43 and pmk-1 in postembryonic nonneuronal cells is essential for CLPr-induced lifespan extension.

To examine whether unc-43 expression in postembryonic neuronal cells is required for CLPr-induced lifespan extension, neuron-specific RNAi experiments were conducted according to the methods of Calixto et al. (35). The sid-1 gene encodes a dsRNA transporter that is necessary for systemic RNAi. Therefore, the lf mutant sid-1(pk3321) does not respond to systemic RNAi induced by feeding. TU3401 worms carry the sid-1(+) transgene controlled by the unc-119 pan-neuronal promoter in the sid-1(pk3321) genetic background. Therefore, systemic RNAi is only effective in neurons in this strain. When the TU3401 worms were fed bacteria expressing unc-43 or pmk-1 dsRNA, the lifespan-extending effect of CLPr was suppressed (Figure 2C, Supplementary Figure 6, Supplementary Table 4). This suggests that CLPr-induced lifespan extension requires the postembryonic expression of unc-43 and pmk-1 in neuronal and nonneuronal cells.

CLPr-Induced Lifespan Extension Is Dependent on Neuronal Factors

To confirm whether the lifespan-extending effect of CLPr is dependent on neurons, we examined whether other neuronal factors are involved in the effect. The odr-3 gene encodes a G-protein alpha subunit that is necessary for chemosensory neuron functions, such as chemotaxis, odorant avoidance, nociception, and cilium morphogenesis (36). This gene is expressed in specific sensory neurons, such as AWC, AWA, AWB, ASH, and ADF neurons, as demonstrated by a reporter gene assay. In particular, the fusion protein ODR-3::GFP is expressed at the highest level in AWC neurons, and is localized to cilia. Worms with the odr-3(n2150) mutation have structural abnormalities in their AWC cilia, and impaired chemotaxis to many volatile odorants. When CLPr was given to worms with the odr-3(n2150) mutation, their lifespan did not increase (Figure 3A, Supplementary Figures 3A and 7, Supplementary Table 5). Furthermore, RNAi of odr-3 in postembryonic TU3401 worms inhibited the CLPr-induced lifespan extension (Figure 2C, Supplementary Figures 3C and 6, Supplementary Table 4). These findings suggest that the postembryonic function of odr-3 in sensory neurons is essential for CLPr-induced lifespan extension.

In AWC neurons, the odr-3 gene product plays a negative role in the cGMP-signaling pathway. This pathway involves guanylyl cyclase and cGMP-gated channel subunits encoded by the odr-1 and tax-4 genes, respectively (37,38). These genes are expressed in sensory neurons, and they are crucial for normal responses to odorants sensed by AWC neurons, and for chemosensation and thermosensation (39). When CLPr was given to worms carrying the lf mutant odr-1(n1933) or tax-4(p678) gene, no lifespan-extending effect was observed (Figure 3A, Supplementary Figures 3A and 7, Supplementary Table 5). This suggests that the cGMP-signaling pathway in sensory neurons is essential for CLPr supplementation-induced lifespan extension. Thus, the genes required for normal AWC function, including unc-43, nsy-1, odr-3, odr-1, and tax-4, are involved in CLPr supplementation-induced lifespan extension.

CLPr-Induced Lifespan Extension is Dependent on AWC Sensory Neurons

We also investigated whether CLPr-induced lifespan extension was dependent on the function of AWC neurons because it was associated with genes essential to their normal function. An experiment was conducted using C elegans strain PY7502 in which the AWC neurons were eliminated via caspase-mediated apoptosis (40). The worms of this strain did not show a significant increase in lifespan upon supplementation with CLPr. This suggests that AWC neurons play a crucial role in CLPr-induced lifespan extension.

AWC neurons form synapses with interneurons AIA, AIB, and AIY. To investigate the involvement of these interneurons in CLPr-induced lifespan extension, the effects of CLPr on the lifespan were examined in 3 different strains, that is, JN578, JN579, and JN580, in which AIB, AIA, and AIY interneurons, respectively, were eliminated by caspase-mediated apoptosis (41). In the JN578 strain, the lifespan-extending effect of CLPr was reduced, indicating that AIB interneurons are involved in CLPr-induced lifespan extension (Figure 3B, Supplementary Figure 8, Supplementary Table 6). In contrast, significant lifespan extension was seen in the JN579 and JN580 strains.

The eat-4 and glr-1 gene products are crucial in synaptic transmission involving glutamate, including the transmission between AWC neurons and their postsynaptic interneurons. Without food, AWC neurons are stimulated to release glutamate from the glutamate transporter EAT-4, which activates AIB neurons via the AMPA-type glutamate receptor GLR-1 (42). CLPr-induced lifespan extension was suppressed in both the lf mutants eat-4(ky5) and glr-1(n2461) (Figure 3A, Supplementary Figure 7, Supplementary Table 5). Although these experiments were only conducted in the presence of FUdR, the results suggest that CLPr-induced lifespan extension depends on the normal functioning of the nervous system.

Active Constituents Responsible for the CLPr-Induced Lifespan Extension

CLPr is a mixture of polyphenols rich in procyanidins with varying degrees of polymerization (Supplementary Figure 1). When the CLPr mixture was separated based on molecular size (30), the high-DP fraction (DP ≥4) at 10 µg/mL was found to have lifespan-extending activity, while the low-DP fraction (DP ≤3) did not (Supplementary Figure 9, Supplementary Table 7). The high-DP fraction-induced lifespan extension was dependent on unc-43 and AWC neurons, but not daf-16. These results suggest that procyanidins with a tetrameric or higher-order structure are involved in the lifespan-extending activity of CLPr. To demonstrate that tetrameric procyanidins have lifespan-extending activity, the tetrameric procyanidin CTA2 was administered to C elegans at a concentration of 10 µg/mL. Supplementation with CTA2 increased the lifespan of wild-type N2, daf-16(mgDf50), and sir-2.1(ok434) worms by 10% but had no significant effect on unc-43(n498n1186) worms or worms in which AWC neurons had been eliminated (Figure 4, Supplementary Figure 10, Supplementary Table 8). These results are consistent with those for CLPr and the high-DP fraction, confirming that at least some tetrameric procyanidins have lifespan-extending activity in C elegans.

CLPr Supplementation Improved Tolerance to Osmotic Stress

CLPr-induced lifespan extension is dependent on postembryonic unc-43 expression in nonneuronal cells as well as the nervous system, as was demonstrated by the RNAi experiments (Figure 2B and C, Supplementary Figures 5 and 6, Supplementary Table 3). Among the genes required for CLPr-induced lifespan extension, osr-1 is expressed in the hypodermis and intestine, and it contributes to survival under osmotic stress through the unc-43/CaMKII and p38 MAPK-signaling cascade (43). To investigate whether CLPr-induced lifespan extension is linked to the osmotic stress response, the effects of CLPr on tolerance to acute and chronic osmotic stress were evaluated.

When exposed to 0.5 M NaCl, C elegans becomes acutely immobile over time. However, when young adult worms were pretreated with 10 μg/mL of CLPr for 24 hours before exposure to 0.5 M NaCl, the time to immobility was longer than that in worms without CLPr pretreatment (Figure 5A). FUdR improved the resistance to acute osmotic stress regardless of whether the worms had been pretreated with CLPr (Figure 5B). The effect of FUdR in improving the resistance to acute osmotic stress was so great that the effect of CLPr might become insignificant.

Subsequently, the impact of CLPr on the survival rate under chronic osmotic stress was investigated without the use of FUdR. Young adult worms were exposed to 0.5 M NaCl in the presence or absence of CLPr at a concentration of 10 µg/mL. After 72 hours, the survival rate was significantly higher among worms with CLPr than among those without CLPr (Figure 5C). The improvement in osmotic tolerance was observed only with the administration of the high-DP fraction, and not the low-DP fraction, similar to the lifespan-extending effects. Moreover, CLPr did not enhance osmotic tolerance in worms with the unc-43(n498n1186) mutation or worms in which the AWC neurons or AIB interneurons had been eliminated (Figure 5D). These findings indicate that CLPr enhances osmotic tolerance through a mechanism similar to that of its lifespan-extending effects.

Increased expression of gpdh-1 is crucial for C elegans to survive under hypertonic stress and to accumulate glycerol as an organic osmolyte (44). After being treated with 10 μg/mL of CLPr for 24 hours, the expression of the GFP reporter gene under the control of the gpdh-1 promoter increased in VP198 worms (Figure 5E and F). The lf mutant gpdh-1(ok1558) worms did not exhibit improved osmotic tolerance with CLPr supplementation (Figure 5D). This suggests that the enhanced osmotic stress tolerance induced by CLPr is dependent on gpdh-1 in the absence of FUdR. However, it is worth noting that CLPr still extended the lifespan of gpdh-1(ok1558) worms (Supplementary Figure 4, Supplementary Table 2). In the presence of FUdR, the CLPr-induced lifespan extension was suppressed in the gpdh-1(ok1558) worms. These results suggest that the mechanism responsible for the CLPr-induced lifespan extension may be related to osmotic tolerance, although the results were not entirely consistent.

Discussion

Comparison of the Prolongevity Effects of CLPr and Other Polyphenols

Procyanidins are oligomeric compounds formed by the condensation of the flavan-3-ols catechin and epicatechin. It has been reported that the lifespan of C elegans can be increased by 100 µM (equivalent to 29 µg/mL) or higher concentrations of monomeric catechin and epicatechin (45,46), and by 65 and 67 µg/mL of oligomeric procyanidin-containing fractions from apple (APr) and blueberry (BPr), respectively (24,25). In the present study, we found that supplementation with CLPr, even at concentrations as low as 10 µg/mL, can increase the lifespan of C elegans (Figure 1, Supplementary Table 1). Comparison of the effects of CLPr to the reported effects of other flavonoids and plant extracts (26) revealed that the effects were similar between CLPr and BPr (24) in many ways. Both CLPr and BPr extended the lifespan in C elegans regardless of whether the worms were fed live bacteria or bacteria killed by ampicillin (BPr) or heat (CLPr), and the lifespan extension was significant at 25°C and 20°C, but not at 15°C (Figure 1, Supplementary Table 1). Most importantly, CLPr and BPr had the same genetic requirements, that is, both showed lifespan-extending activity in sir-2.1, daf-16, or skn-1 lf mutants, but not in osr-1, unc-43, sek-1, or mev-1 lf mutants (Figure 2, Supplementary Figures 3 and 4, Supplementary Table 2). These consistencies suggest that CLPr and BPr share a common mechanism for extending the lifespan of C elegans.

Regarding the effects of APr, APr differed from CLPr and BPr in that the extension of lifespan induced by 65 µg/mL of APr was dependent on sir-2.1 (25). On the other hand, regarding the effects of cocoa powder, even though it is derived from the same plant species as CLPr, 4 mg/mL cocoa powder was found to extend the lifespan of C elegans in a manner that was dependent on sir-2.1 and daf-16 (47), unlike CLPr. This concentration of cocoa powder was 400 times greater than the concentration of CLPr (10 µg/mL) used in the present study. The differences in the lifespan-extending effects between CLPr and APr or cocoa powder may be because the active constituents are different.

Procyanidins With a Tetrameric or Higher-Order Structure Are the Active Constituents Responsible for CLPr-Induced Lifespan Extension

The high-DP fraction-induced lifespan extension, whereas the low-DP fraction did not (Supplementary Figure 9, Supplementary Table 7), indicating that neither catechin nor epicatechin was responsible for the lifespan extension induced by 10 µg/mL of CLPr. This is also evidenced by the differences in the genetic requirements for the lifespan-extending effects between catechin and CLPr; catechin does not require osr-1, unc-43, or sek-1 (45). Instead, procyanidins with a tetrameric or higher-order oligomeric structure are thought to be the active constituents of CLPr. This is supported by the findings that similar to CLPr, tetrameric CTA2 and the high-DP (DP ≥ 4) fraction-induced lifespan extension in a manner that was dependent on unc-43 and AWC neurons, but not daf-16 or sir-2 (Figure 4, Supplementary Figures 9 and 10, Supplementary Tables 7 and 8).

The Nervous System Is Essential for Procyanidin-Induced Lifespan Extension

Although many studies have shown that polyphenols can extend the lifespan of C elegans, the effects of the polyphenols on the nervous system have remained unknown. However, the findings of the present study demonstrated that normal nervous system functioning is vital for CLPr-induced lifespan extension, as follows:

• (1) CLPr-induced lifespan extension required neuronal expression of unc-43, pmk-1, and odr-3 during the postembryonic stages, as demonstrated by the RNAi experiments in TU3401 worms (Figure 2C, Supplementary Figures 3C and 6, Supplementary Table 4).

• (2) CLPr did not extend the lifespan of worms with a lf mutation in the odr-3, odr-1, or tax-4 genes that are necessary for the functions of sensory neurons, including AWC neurons (Figure 3A, Supplementary Figures 3A and 7, Supplementary Table 5). The odr-3 gene is expressed in specific sensory neurons, with the highest expression in AWC neurons (36).

• (3) CLPr did not extend the lifespan of worms in which AWC neurons had been eliminated (Figure 3B, Supplementary Figure 8, Supplementary Table 6).

• (4) CLPr did not significantly extend the lifespan of worms in which AIB interneurons, one of the postsynaptic interneurons of AWC neurons were eliminated (Figure 3B, Supplementary Figure 8, Supplementary Table 6).

• (5) CLPr-induced lifespan extension required the eat-4 and glr-1 genes, which encode a glutamate transporter and receptor, respectively, involved in synaptic transmission from AWC neurons to AIB interneurons (Figure 3A, Supplementary Figure 7, Supplementary Table 5).

In addition, the high-DP fraction and CTA2, like CLPr, extended the lifespan in a manner that was dependent on AWC neurons (Figure 4, Supplementary Figures 9 and 10, Supplementary Tables 7 and 8). Thus, the nervous system, including AWC neurons, is essential for the lifespan-extending effects of procyanidins in C elegans.

Mechanistic Relationship Between the CLPr-Induced Lifespan Extension and the Improvement of Osmotic Stress Tolerance

The CLPr-induced lifespan extension was found to be dependent on nonneuronal unc-43 and pmk-1, as well as on hypodermal and intestinal osr-1 (43) (Figure 2, Supplementary Figures 3 and 4, Supplementary Tables 2 and 3). The osr-1 gene is expressed in the hypodermis and intestine and negatively regulates physiological and behavioral responses to hyperosmotic environments (43). In the osmotic response, OSR-1 has been suggested to function upstream or parallel to the UNC-43/CaMKII and NSY-1/SEK-1/PMK-1 p38 MAPK pathway. All of these factors play a crucial role in CLPr-induced lifespan extension, suggesting that CLPr may increase the lifespan through a mechanism related to the osmotic response. Consistent with this, supplementation with CLPr or the high-DP fraction increased the osmotic tolerance of C elegans in a manner that was dependent on AWC neurons, AIB interneurons, and unc-43 (Figure 5). Although the osmotic response has been reported to be dependent on ASH sensory neurons (48), there has been no evidence linking it to AWC neurons. In the present study, we showed that the enhanced osmotic stress tolerance induced by CLPr is dependent on AWC neurons and AIB interneurons.

The gpdh-1 gene encodes an osmolyte biosynthesis enzyme glycerol-3-phosphate dehydrogenase that is expressed in the hypodermis and intestine, and is essential for osmotic responses (43). CLPr appears to enhance osmotic tolerance by inducing the expression of gpdh-1, because CLPr treatment increased the expression of the gpdh-1_P::GFP transgene, and the CLPr-induced improvement in osmotic stress tolerance was suppressed in the gpdh-1 lf mutant (Figure 5D and F). CLPr supplementation may enhance osmotic tolerance by suppressing osr-1, a negative regulator of gpdh-1 expression (43).

In the gpdh-1(ok1558) lf mutant, CLPr did not improve the tolerance to chronic osmotic stress, but it did increase the lifespan of C elegans in the absence of FUdR (Figure 5D, Supplementary Figure 4, Supplementary Table 2). This seems consistent with the description by Dues et al., who stated that although stress resistance and lifespan extension share common genetic pathways, stress resistance is not always correlated with longevity, and can be separated experimentally (49). Although it can be inferred that CLPr-induced lifespan extension and osmotic stress tolerance are likely related, other factors besides gpdh-1 may also be involved in lifespan extension. For example, in gpdh-1(ok1558) mutants, intact gpdh-2 may extend the lifespan in the absence of FUdR by compensating for the gpdh-1 deficiency, but it does not improve the stress tolerance to chronic osmotic stress.

As Anderson et al. reported (50), FUdR increased the resistance to acute hypertonic stress (Figure 5B). In the presence of FUdR, CLPr supplementation did not confer additional resistance to acute osmotic stress. However, even in the presence of FUdR, CLPr was able to increase the lifespan in a gpdh-1-dependent manner (Figure 1C, Supplementary Figure 4, Supplementary Tables 1 and 2). This suggests that the mechanisms behind CLPr-induced lifespan extension and enhanced osmotic stress tolerance are related, but can be separated depending on the experimental conditions. Again, in addition to gpdh-1, other factors may also contribute to this process.

Consideration of the Mechanism by Which Procyanidins With a Tetrameric or Higher-Order Multimeric Structure Act Through AWC Neurons

This study found that the ability of CLPr to improve osmotic stress tolerance and extend the lifespan was due to procyanidins with a tetrameric or higher-order polymeric structure, which have molecular weights exceeding 1 000 g/mol (Figures 4 and 5, Supplementary Figures 9 and 10, Supplementary Tables 7 and 8). These effects were dependent on AWC neurons and several nonneuronal factors (Figures 2 and 3B, Supplementary Figures 3, 4, and 8, Supplementary Tables 2 and 6). One of the nonneuronal factors was osr-1, the expression of which in the hypodermis is essential for osmotic responses (43). However, because neither AWC neurons nor the hypodermis is exposed to the external environment (48), it is unlikely that procyanidins with a molecular weight of over 1 000 g/mol can directly affect AWC neurons or the hypodermis through the cuticle layer. This raises the question of how procyanidins can affect AWC neurons or the hypodermis.

Procyanidins are a type of tannin with astringent properties. Therefore, they may adhere to the cuticle or the intestinal lining, and act like an osmotic stimulus. Alternatively, it is possible that procyanidins may affect AWC neurons indirectly through other neurons, such as gustatory neurons, that penetrate the cuticle and expose their sensory cilia to the environment (48). Further research is needed to elucidate the mechanisms by which procyanidins affect the nervous system.

Conclusions and Perspectives

This study revealed that procyanidins with a tetrameric or higher-order structure can increase the lifespan of C elegans. Although it was unknown whether the nervous system is involved in the lifespan-extending effect of polyphenols, such as procyanidins, we found that the lifespan-extending effect was dependent on the nervous system and the unc-43/CaMKII-p38 MAPK pathway. Although this study was conducted using C elegans, our results may provide new insights into the effects of procyanidins in humans. Several recent studies have suggested that the tetrameric procyanidin CTA2 has various physical activities in mice and rats, some of which are related to the nervous system. For the utilization of procyanidins to promote health, it is essential to understand how procyanidins with a molecular weight of over 1 000 g/mol affect the nervous system. Investigations on the mechanism by which procyanidins extend the lifespan of C elegans through the nervous system are expected to provide valuable insights.

Funding

This work was supported by JSPS KAKENHI [grant numbers 20611017 and 21K06618] and partially by Meiji Co. Ltd. in 2010. Most strains used were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).

Conflict of Interest

None.

Acknowledgment

The authors would like to thank Professor Yuichi Iino at the University of Tokyo for providing us with the JN578 strain. The authors also thank Dr. Yon-Suk Yun, Mr. Kosuke Amagasa, Ms. Asuka Kambe, Ms. Ayaka Sasho, Mr. Kanta Ikenoue, Mr. Rikuto Shimizu, Mr. Takahikro Fujimura, and Mr. Fumiya Katsumata for their collaboration.

Author Contributions

H.I. designed and conceptualized the study and wrote the manuscript. K.O. analyzed the data and wrote a part of the manuscript. Y.M., S.K., A.M., K.S., S.T., H.S., and H.H. performed lifespan assay and other experiments and analyzed the data. Y.F. provided technical advice on imaging analysis, and K.S. and M.N. provided technical support for the extraction of procyanidins from cacao. All authors had access to the data, contributed to the interpretation of findings, and revised and approved the final manuscript.

References