1 
Montmorency tart cherry (Prunus cerasus L.) acts as a calorie restriction 1 
mimetic that increases intestinal fat and lifespan in Caenorhabditis elegans 2 
 3 
David van de Klashorst1, Amber van den Elzen1, Jasper Weeteling1, Michael Roberts2, 4 
Terun Desai2, Lindsay Bottoms2 and Samantha Hughes1*  5 
 6 
1HAN BioCentre, HAN University of Applied Sciences, Nijmegen, The Netherlands 7 
2Centre for Research in Psychology and Sports Science, University of Hertfordshire, 8 
Hertfordshire, UK 9 
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* corresponding author: samantha.hughes@han.nl 11 
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Short title: Montmorency tart cherries provides health promoting effects in C. elegans. 13 
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Abstract: 15 
Montmorency Tart Cherries, MTC, (Prunus cerasus L.) possess a high anthocyanin 16 
content as well as one of the highest oxygen radical absorbance capacities fruits at 17 
common habitual portion sizes. MTC have been shown to contribute to reducing 18 
plasma lipids, plasma glucose and fat mass in rats and strikingly, similar effects are 19 
observed in humans. However, there is a paucity of research examining the molecular 20 
mechanisms by which such MTC effects are induced. Here, we show that when 21 
exposed to MTC, Caenorhabditis elegans display an extension of lifespan, with a 22 
corresponding increase in fat content and increase in neuromuscular function. Using 23 
RNA interference, we have confirmed that MTC is likely to function via the Peroxisome 24 
Proliferator-Activated Receptor (PPAR) signalling pathway. Further, consumption of 25 
 2 
MTC alters the pharyngeal pumping rate of worms which provides encouraging 26 
evidence that MTC may be operating as a calorie restriction mimetic via metabolic 27 
pathways. 28 
 29 
Key words:  30 
Healthy aging; pharyngeal pumping; Metabolic Syndrome; lifespan; polyphenol; 31 
anthocyanins 32 
 33 
Highlights: 34 
• Montmorency tart cherries increases lifespan and fat content in low doses  35 
• MTC reduces pumping rate in C. elegans suggesting it acts as a calorific 36 
mimetic 37 
• MTC acts via the PPAR pathway in C. elegans, as it does in other systems 38 
• Low doses of MTC have a positive effect on healthy aging of C. elegans  39 
 40 
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1. Introduction 42 
Modern dietary habits result in a myriad of cardio-metabolic dysfunction leading to the 43 
development of metabolic Syndrome (MetS), and eventually chronic disease. It is 44 
estimated that, worldwide, one quarter of the world population, estimated to be over 1 45 
billion people have Metabolic Syndrome (MetS) (Saklayen, 2018), a cluster of cardio-46 
metabolic criteria including obesity, hyperglycaemia, dyslipidaemia and elevated blood 47 
pressure (Holubkova, Penesova, Sturdik, Mosovska, & Mikusova, 2012; Saklayen, 48 
2018). MetS is often a precursor to type 2 diabetes and cardiovascular disease, which 49 
together place a significant burden on health services and are the leading causes of 50 
reduced lifespan and morbidity worldwide (O'Neill, Bohl, Gergersen, Hermansen, & 51 
 3 
O'Driscoll, 2016). Given the social challenges faced by the prevalence of MetS, 52 
obesity, cardiovascular disease and diabetes, non-pharmacological interventions are 53 
desperately needed to safely prevent and mitigate the development of these diseases.  54 
 55 
Recently, there has been renewed interest into diets supplemented with “functional 56 
foods” particularly rich in polyphenols for health and exercise benefits, namely beetroot 57 
juice (Ferreira & Behnke, 2010), purple sweet potatoes (Chang, Hu, Huang, Yeh, & 58 
Liu, 2010), blueberries (McAnulty et al., 2011; Wilson et al., 2006), pomegranate juice 59 
(Thrombold, Reinfeld, Casler, & Coyle, 2011), pitanga fruit (Tambara et al., 2018), 60 
green tea (Jowko et al., 2011) and cherries (Bell, McHugh, Stevenson, & Howatson, 61 
2014; Traustadottir et al., 2009). Ensuring a diet rich in such foods results in significant 62 
health benefits to humans, specifically related to their antioxidative, anti-inflammatory, 63 
anti-obesity and anti-cancer properties (Ghosh, 2005; Seymour et al., 2009; Wang & 64 
Stoner, 2008; Wu et al., 2006). Montmorency Tart Cherries (Prunus cerasus L.), MTC, 65 
possess a high anthocyanin content and has one of the highest oxygen radical 66 
absorbance capacities of fruits consumed at common habitual portion sizes (Ou, 67 
Bosak, Brickner, Iezzoni, & Seymour, 2012). The health benefit of MTC is likely due to 68 
the presence of polyphenols, mainly anthocyanins, which are commonly found in the 69 
skin of the fruit and are responsible for its dark red pigment (Khoo, Azlan, Tang, & Lim, 70 
2017). 71 
 72 
Human studies have established that MTC has anti-inflammatory (Bell, McHugh, et al., 73 
2014), antioxidative (Bell, McHugh, et al., 2014), anti-hypertensive (Keane, George, et 74 
al., 2016) and anti-hyperuricaemic (Bell, Gaze, et al., 2014) properties. 75 
Correspondingly, rats fed MTC displayed significantly improved lipid profiles and 76 
 4 
reduced fat mass, hyperinsulinaemia and hyperglycaemia compared to control animals 77 
(Seymour et al., 2009; Seymour et al., 2008). Additionally, tart cherries have been 78 
shown to have an anti-diabetic effect in diabetic rats, via the reduction in plasma 79 
glucose (Tahsini & Heydari, 2012). Together, although this is promising evidence that 80 
MTC can thwart MetS development, there is a paucity of research examining 81 
mechanisms of action by which these effects are induced. Previous studies 82 
investigating the mechanisms underpinning MTC used animal models, and showed 83 
that MTC induced gene expression of peroxisome proliferator-activated receptors 84 
(PPARα/γ) and downregulated IL-6 (interleukin-6) and TNF-α (tumour necrosis factor-85 
alpha) (Seymour et al., 2009; Seymour et al., 2008), all major pathways involved in fat 86 
metabolism and insulin signalling. It is likely that the phytochemicals in MTC may alter 87 
the transcription of other genes involved in the response to oxidative stress 88 
(Kirakosyan, Gutierrez, Solano, Seymour, & Bolling, 2018), but these have yet to be 89 
fully elucidated. It is therefore obvious that a detailed understanding of the effect of 90 
MTC on these, and other, pathways that lead to positive health effects is lacking.  91 
 92 
We have used the model organism Caenorhabditis elegans to study the positive health 93 
benefits provided by MTC. C. elegans is a powerful model with which to study the 94 
molecular pathways that underpin human disease (Markaki & Tavernarakis, 2010; 95 
Shaye & Greenwald, 2011b) but also with which to investigate functional foods. C. 96 
elegans is a small, transparent nematode worm with many well-studied molecular 97 
networks and tissue systems that are also found in vertebrates (Kaletta & Hengartner, 98 
2006; Lehner, Crombie, Tischler, Fortunato, & Fraser, 1996; Shaye & Greenwald, 99 
2011a; Sulston & Horvitz, 1977). The nematode has been fully sequenced and 100 
mapped (Hiller et al., 2005), with 80% of human genes possessing homologs in C. 101 
 5 
elegans (Kaletta & Hengartner, 2006). In addition, feeding behaviour, nutritional 102 
uptake and fat metabolism are conserved between C. elegans and humans (Hasmi et 103 
al., 2013). Importantly, C. elegans is frequently used to investigate the biological 104 
functions of food compounds (Buchter et al., 2013; Chen, Muller, Richling, & Wink, 105 
2013; Gao et al., 2015; Grunz et al., 2012; Kock, Weldle, Baler, Buchter, & Watjen, 106 
2019; Tambara et al., 2018; Wilson et al., 2006). Taken together, these factors 107 
emphasise the translational relevance of this model species and its potential to predict 108 
effects in higher animals and inform clinical nutrition practice in humans. 109 
 110 
We have chosen to use C. elegans to better understand the molecular basis of the 111 
health promoting effects of MTC by examining the response of C. elegans to various 112 
dilutions of MTC concentrate. We hypothesised that exposure to MTC will increase 113 
lifespan and reduce fat content. We also used RNAi to silence genes involved in fat 114 
metabolism, conserved between humans and nematodes, to provide insight into the 115 
molecular pathways by which MTC acts. Lastly, using pharyngeal pumping assays, we 116 
provide evidence that MTC does act as a calorie restriction mimetic.  117 
 118 
2. Materials and Methods 119 
2.1 Strains and maintenance of worms 120 
Strains used in this study (wild type N2 var. Bristol and TJ356 [zIs356 IV(pdaf-16::daf-121 
16::gfp; rol-6)]) and bacterial strains were provided by the Caenorhabditis Genetics 122 
Centre (CGC). C. elegans strains were maintained on Nematode Growth Media 123 
(NGM) agar  prepared according to standard protocols (Brenner, 1974) and plates 124 
seeded with OP50 E. coli as a bacterial food source. 125 
 126 
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To synchronise worm populations, gravid worms were washed from plates with M9 127 
buffer (Brenner, 1974) and bleached according to standard protocols (Stiernagle, 128 
2006) using alkaline hypochlorite solution (4ml 5% sodium hypochlorite, 1ml 4M 129 
sodium hydroxide, 5ml dH2O). Released eggs were left to hatch overnight at room 130 
temperature (19-20°C) in M9 buffer in the absence of a food source, giving rise to a 131 
population of synchronised L1 larvae which could then be placed directly onto NGM 132 
to develop at a similar rate. 133 
 134 
2.2 MTC concentrate seeded NGM  135 
Concentrated Montmorency Tart Cherry juice was obtained from Cherry Active (now 136 
called Active Edge, 5-060142-250010, Hanwell, UK). This product is 100% 137 
concentrated Montmorency Cherry juice with no added preservatives. Cherry Active 138 
suggests that the recommended daily human consumption is 30ml MTC concentrate 139 
mixed with 240ml water. Total phenolic content of Cherry Active MTC was determined 140 
to be 726mg/ml by the Folin-Ciocalteu assay (Swain & Hillis, 1959) using a gallic acid 141 
standard curve.  142 
 143 
Concentrated MTC Juice (Cherry Active) was added directly to the cooled molten 144 
NGM prior to pouring plates. The final concentrations of MTC juice in the NGM plates 145 
ranged from 16.7µl/ml to 150µl/ml in NGM. The pH of all preparations was checked, 146 
substituting water for NGM. For each MTC spiked water sample, there was no change 147 
in pH compared to the control. 148 
 149 
2.3 Development assay 150 
 7 
Two assays were undertaken, both adapted from Xiong et al. (Xiong, Pears, & 151 
Woollard, 2017). The first was a simple lawn clearance based where single L4 animals 152 
were placed on 2ml NGM, with or without MTC, in a 12-well plate containing 30µl 153 
OP50. This was classed as day 0. The plates were monitored daily for clearance of 154 
the bacterial lawn. In the second assay, L1 animals were added to plates, prepared in 155 
the same way as previously, and each day the worms were observed for development. 156 
The addition of L1 animals to the plates was at time 0. Worms were scored as younger 157 
than L4 (<L4), L4 or egg laying. Worms that were dead were also scored. 158 
 159 
2.4 RNA interference 160 
RNA interference experiments were performed using standard protocols (Kamath & 161 
Ahringer, 2003). NGM for RNAi was supplemented with 116.7µl/ml of MTC 162 
concentrate in NGM, or an equivalent volume of distilled water as the control. The 163 
RNAi clones were chosen based on their homology to human genes involved in 164 
different aspects of fat metabolism (Ashrafi et al., 2003; Shaye & Greenwald, 2011b). 165 
Clones were obtained from the Vidal ORFeome-based RNAi library (Rual et al., 2004) 166 
and those used in this research are listed in Table 1. All nematode experiments were 167 
conducted using the N2 strain. dsRNA was delivered by feeding to age-synchronised 168 
L1 stage animals, which were incubated at 20oC and phenotypes were observed 48 169 
hours later, when control animals had reached the L4 stage. Control RNAi was 170 
performed using HT115 bacteria transformed with an empty L4440 vector.  171 
 172 
2.4 Lifespan assay 173 
To assess lifespan, we used wild type N2. Animals were reared on OP50 seeded NGM 174 
at 16oC following standard protocols. When gravid, animals were bleached and the 175 
 8 
resulting age synchronised L1 animals were placed onto seeded NGM. When animals 176 
were L4, this was counted as Day 0 of the survival assay. During the reproductive 177 
period, the worms were transferred to a new plate and observed every day for death. 178 
Animals were scored as dead if they failed to respond to a touch by a platinum wire. 179 
Survival curves were generated and data analysed using the OASIS software (Yang 180 
et al., 2011). For multiple analysis, p<0.0055 was considered as significant (Bonferroni 181 
corrections).  182 
 183 
2.5 Obesity assay 184 
The obesity assay was adapted from Mak et al. (Mak, Nelson, Basson, Johnson, & 185 
Ruvkun, 2006) and Ashrafi et al. (Ashrafi et al., 2003). A stock solution of Nile Red 186 
(Roth) was prepared at 0.5mg/ml in acetone and diluted in 1xPBS to 1µg/ml, both 187 
solutions were stored in the dark at 4oC. Nile Red was added to molten NGM (1ml of 188 
working solution in 50ml NGM) and when solid the plates were seeded with E. coli 189 
OP50. Age synchronised L1 worms were added to the plates and allowed to develop 190 
to L4, at which point the worms (n=26 per condition) were sacrificed for microscopy. 191 
 192 
Individual worms were mounted onto 2% agarose pads in 0.1% sodium azide. 193 
Fluorescent imaging was carried out using a Zeiss Imager.M2 microscope and 194 
photomicrographs were taken using a x63 objective (Zeiss) and Zeiss Zen 2012 Blue 195 
Software. Images were taken using the same gain and exposure settings of animals 196 
on the same focal plane (at the grinder in the second pharyngeal bulb). Using ImageJ 197 
software, the fluorescence in a 20x20 pixel box placed at the grinder was calculated, 198 
to give a value corresponding to level of staining of the Nile Red vital dye. The arbitrary 199 
values for “red” are equivalent to the level of Nile Red staining, which are normalised 200 
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to the control worms and plotted with standard deviation error bars. The data is 201 
analysed using the t-test.  202 
 203 
2.6 Microscopy 204 
DIC images were taken using a Leica benchtop M165FC microscope equipped with a 205 
Leica DFC425C camera and photomicrographs taken using LAS V4.2 software. To 206 
take fluorescent images, worms were mounted onto 2% agarose pads in 0.1% sodium 207 
azide. Fluorescent imaging was carried out using a Zeiss Imager.M2 microscope and 208 
images taken using Zeiss Zen 2012 Blue Software. All images were compiled using 209 
Adobe Photoshop 7.0 and backgrounds merged.  210 
 211 
2.7 Pharyngeal pumping 212 
Gravid animals were subjected to sodium hypochloride treatment, and synchronised 213 
L1 nematodes placed directly onto NGM in the presence or absence of MTC (at either 214 
16.7µl/ml or 66.7µl/ml). When animals had reached L4 stage, worms were washed off 215 
the plates with M9 buffer. After removal of the bacteria, worms were resuspended in 216 
M9 and serotonin (10mM final concentration) to stimulate pumping. After 10 minutes 217 
incubation in serotonin, electropharyngeogram recordings were taken using the 218 
NemaMetrix ScreenChip system using an SC30 chip. Each recording was 2 minutes 219 
in duration and analysed using the NemaMetrix ScreenChip software in 2 independent 220 
experiments. The data was combined for each MTC condition and the averages and 221 
standard error of the mean plotted for pump frequency, duration, inter pump interval 222 
and amplitude with a t-test used for statistical analysis.  223 
   224 
2.8 Localisation of DAF-16::GFP 225 
 10 
Intracellular localisation of DAF-16 was observed using the strain TJ356. 226 
Synchronised L1 animals were placed on MTC spiked NGM and allowed to develop 227 
to L4. At this point, 100 animals were assessed for DAF-16::GFP localisation using a 228 
Leica M165FC microscope, based on the method described in Büchter et al (Buchter 229 
et al., 2013). 230 
 231 
2.9 Pluronic gel mobility assay 232 
Pluronic F-127 is a biocompatible thermoreversable hydrogel which is a favourable 233 
environment in which to perform a burrowing assay (Lesanpezeshki et al., 2019). 234 
Worms are age synchronized and grown to L4 stage, at which point they are 235 
transferred to NGM spiked with MTC (to 16.7µl/ml and 66.7µl/ml) and FuDR at a final 236 
concentration of 10mM. The plates are incubated at 20oC for 1 week at which point 237 
the worms are used for the pluronic gel assay. 238 
 239 
A gel concentration of 26% (w/w with distilled water) is prepared and a droplet added 240 
to the bottom of a 12-well plate. Worms (wild type N2) are added to the droplet, and 241 
then pluronic gel added up to a height of 700mm. To the surface of the well, OP50 and 242 
1% isoamyl alcohol (25µl) is added, when time is recorded as time 0. The wells are 243 
monitored every 15 minutes over a 2 hour period, and the number of worms at the 244 
surface of the well counted. Each experiment is performed in triplicate with 30 worms 245 
in 2 independent experiments.  246 
 247 
3. Results and discussion 248 
3.1 Relating human dose to C. elegans 249 
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Various human studies have investigated the effect of MTC on humans, however there 250 
is very little coherence in MTC dosing between studies. The lowest dilution used in a 251 
trial is 30ml MTC with 220ml water, equivalent to 120µl MTC/ml water, henceforth 252 
described as 120µl/ml (Lynn et al., 2014), while other studies use 30ml MTC in 100ml 253 
water (230µl/ml) (Desai, Bottoms, & Roberts, 2018; Desai, Roberts, & Bottoms, 2019) 254 
up to a maximal dilution of 60ml MTC in 100ml water (375µl/ml) (Keane, George, et 255 
al., 2016; Keane, Haskell-Ramsey, Veasey, & Howatson, 2016). To further compound 256 
the issue, the recommended daily dose as prescribed by Cherry Active is where 30ml 257 
MTC is mixed with 240ml water (equivalent to 111µl/ml). Moreover, despite 258 
pharmacokinetic studies with MTC examining anthocyanin and phenolic 259 
concentrations in systemic circulation (Bell, Gaze, et al., 2014; Keane, Bell, et al., 260 
2016; Seymour et al., 2014), there is no evidence of how much MTC is being absorbed 261 
into tissues in humans, although this has been investigated in healthy rats (Kirakosyan 262 
et al., 2015). 263 
  264 
Therefore, to try to equate these human consumption doses to C. elegans, a dose 265 
response curve was undertaken using development as an end point. MTC 266 
concentrations ranged from 16µl/ml to 300µl/ml, however, worms did not survive on 267 
MTC concentrations above 150µl/ml (data not shown) and so these were not tested 268 
further. Nematodes exposed to low dilutions of MTC developed at a similar rate to 269 
controls, until the MTC concentration was 66µl/ml or more (Figure 1). At 66.7µl/ml 270 
MTC, the nematodes displayed a delayed development compared to control animals, 271 
but did reach the egg laying stage. In contrast, few animals were able to reach egg 272 
laying age when exposed to 100µl/ml MTC or more (Figure 1). For this reason, we 273 
chose to focus our efforts on those MTC dilutions that were relevant for C. elegans 274 
(i.e. up to 100µl/ml). In this way, it is possible to comment on the general health 275 
promoting benefits of MTC. However, this does highlight a problem when linking the 276 
 12 
data observed in C. elegans, and other model organisms. However, investigating the 277 
general health promoting effects of MTC is valuable using C. elegans as it provides 278 
an indication of the possible mechanistic basis for the health promoting effects (Desai 279 
et al., 2018; Desai et al., 2019; Seymour et al., 2014). 280 
 281 
3.2 Lifespan is increased following exposure to MTC 282 
Studies have shown that longevity is related to a metabolic shift away from glucose 283 
metabolism towards lipid oxidation (van Heemst, 2010). Therefore, we were interested 284 
to see if exposure to MTC has any effect on lifespan. Wild type animals that were not 285 
exposed to MTC lived for a maximum of 17 days post L4, while animals exposed to 286 
MTC displayed a small but significant increase in lifespan (Figure 1, Table 2). This 287 
extension to lifespan was most striking at lower MTC dilutions, 16.7µl/ml of MTC, the 288 
longest-lived animal was 23 days old and at 150µl/ml, the maximum lifespan was 20 289 
days (Figure 1, Table 2). Intriguingly, the mean lifespan was only extended for 290 
nematodes exposed to the lower doses of MTC (16.7µl/ml), with higher levels of MTC 291 
having a mean lifespan less than that of control animals.  292 
 293 
Together, our data shows that, at least concerning lifespan, lower dilutions of MTC are 294 
more beneficial to the worms than higher dilutions. It is possible that a hormetic effect 295 
of MTC is occurring, where the benefit provided by lower levels of anthocyanins, the 296 
main polyphenols in MTC,  is to augment endogenous antioxidant capacity and  297 
scavenge free radicals more effectively, while at higher concentrations the pro-oxidant 298 
nature of the anthocyanins reverses some health-promoting effects (Blando, Gerardi, 299 
& Nicoletti, 2004; Konczak & Zhang, 2004).  300 
 301 
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Such a lifespan extension as observed here is similar to that of blueberry polyphenols, 302 
which were able to increase lifespan and delay aging in C. elegans (Wilson et al., 303 
2006). This may be related to the fact that despite tart cherries containing sugar, which 304 
would be expected to reduce lifespan, the fruit is able to reduce glycaemic stress by 305 
modulating the insulin signalling pathway (Seymour et al., 2009). Such a reduction in 306 
glycaemic stress may be mediated by the presence of MTC anthocyanins, phenolic 307 
acids and/or their secondary metabolites (Kirakosyan, Seymour, Urcuyo-Lianes, 308 
Kaufman, & Bolling, 2009; Scazzocchio et al., 2011), which may reduce the 309 
concentration of ligands available to activate the insulin signalling cascade.  310 
 311 
3.3 Lifespan extension by MTC exposure is not mediated by DAF-16/FoxO 312 
To assess if the lifespan extension provided by MTC is a result of DAF-16 signalling, 313 
we observed the localisation of DAF-16. Our results show that MTC did not have a 314 
striking effect on DAF-16::GFP localisation at all MTC concentrations tested, with 315 
localisation being predominantly cytoplasmic (Figure 3). This suggests that the MTC 316 
is not activating stress response genes and it is also unlikely that the lifespan extending 317 
effects are due to activation of the insulin signalling pathway. 318 
 319 
3.4 MTC acts though the PPAR signalling pathway 320 
As there is significant homology between the mechanisms of fat storage and regulation 321 
in C. elegans and mammals (Ashrafi et al., 2003; Hashmi et al., 2013), it is possible to 322 
unravel the pathways through which MTC acts. We chose a number of candidate 323 
genes based on their homology to key human fat metabolism processes to silence by 324 
RNAi (Table 1). During early experiments, a significant developmental delay at L4 was 325 
noted at MTC concentrations above 100µl/ml. Thus, the genes of interest were 326 
 14 
silenced and after 3 days at 20oC, the developmental stage of animals were compared 327 
to control worms which had reached L4. 328 
 329 
The majority of the genes tested showed a similar effect to the control RNAi, L4440, in 330 
the presence of MTC, in that all animals reached the L4 stage at approximately the 331 
same time. However, 7 genes that were silenced in the presence of MTC showed a 332 
similar stage of development as the control in the absence of MTC i.e. a recovery of 333 
the developmental delay and a further 7 resulted in an enhancement of the 334 
developmental delay caused by MTC alone (Table 1). 335 
 336 
RNAi knockdown of daf-22 in the presence of MTC reversed the developmental delay 337 
that is caused by exposure to MTC, while on the other hand knockdown of nhr-49 338 
further enhanced the developmental delay compared to the control. Such an effect is 339 
intriguing, as both daf-22 (nematode homolog of human SCP2) and nhr-49 (the 340 
HNF4G/PPARa homolog) function in the PPAR (peroxisome proliferator-activated 341 
receptor) signalling pathway (Dansen et al., 2004; van Gilst, Hadjivassiliou, Jolly, & 342 
Yamamoto, 2005), which are central regulators of fat metabolism (Nunn, Bell, & Barter, 343 
2007). Therefore, we are able to confirm that MTC influences the PPAR pathway, as 344 
shown previously (Seymour et al., 2008) and thus is likely to operate as a calorie 345 
restriction mimetic (Corton et al., 2004). 346 
 347 
It is striking that when sbp-1 (the nematode homolog of SREBP1c, Sterol regulatory 348 
element binding protein) is silenced in the presence of elevated MTC dilutions there is 349 
a recovery of the developmental delay. In contrast, there is an enhancement of the 350 
MTC induced developmental delay when lipl-1 (a lysosomal lipase) is knocked down. 351 
 15 
Both sbp-1 and lipl-1 have recently been shown to be regulated by the MAX-3 352 
transcription factor, to modulate lipid metabolism in nematodes (Mejia-Martinez et al., 353 
2017). It is likely, that these genes form part of a complex network modulating the 354 
response to carbohydrates (including glucose) and fat, as suggested previously.  355 
 356 
Ultimately, regulation of longevity and lipid metabolism is complex, involving multiple 357 
genes and signalling pathways and how longevity and fat accumulation are uncoupled 358 
from diet remains elusive. To elucidate the complete mechanism behind the positive 359 
health benefits of MTC is beyond the scope of this work, but will provide a wealth of 360 
information linking various aspects of diet, lifespan and health which will have 361 
significant impact on the dietary advice given to humans suffering with MetS.  362 
 363 
3.4 Exposure to high MTC dilutions enhances lipid staining 364 
Fat metabolism and lifespan are intertwined and likely to be directly coupled (Hansen, 365 
Flatt, & Aguilaniu, 2013). To explore the effect of MTC on fat accumulation, we 366 
therefore  measured the fat content of MTC exposed animals using the vital dye, Nile 367 
Red (Ashrafi et al., 2003; Mak et al., 2006). Strikingly, animals exposed to the lower 368 
doses of MTC (16.7µl/ml and 66.7µl/ml) displayed a significantly increased level of Nile 369 
Red staining compared to controls (Figure 4). In contrast, elevated levels of MTC 370 
significantly reduced Nile Red staining (83.3µl/ml).  371 
 372 
Similar to our results, other long-lived mutants display an increase in fat accumulation 373 
(O'Rourke, Soukas, Carr, & Ruvkin, 2009).  Although MTC has an elevated 374 
carbohydrate content, this would prompt the activation of transcription factors that 375 
promote the expression of antioxidant or detoxification enzymes (Pang, Lynn, Lo, 376 
 16 
Paek, & Curran, 2014), thus allowing animals to promote longevity, as shown 377 
previously in blueberries (Wilson et al., 2006), supporting our hypothesis for the 378 
hormetic action of MTC anthocyanins. As lipids are more energy-dense than 379 
carbohydrates, the storage of lipids provides a survival advantage under calorie 380 
restriction conditions (van Heemst, 2010). 381 
 382 
3.5 Pumping 383 
Moderate dietary restriction in C. elegans is usually achieved by reducing food intake 384 
by altering the pharyngeal pumping rates (Avery, 1993) and such dietary restriction is 385 
able to extend lifespan (Greer & Brunet, 2009; Lakowski & Hekimi, 1998). We 386 
measured the pharyngeal pumping rate of L4 stage C. elegans grown in the presence 387 
or absence of MTC (at 16.7µl/ml and 66.7µl/ml).  We found that the pumping frequency 388 
was reduced following exposure to MTC (Figure 5). Strikingly, the pump duration, 389 
interpump interval and amplitude was increased in a dose dependent manner when 390 
worms were exposed to MTC (Figure 5). Together this suggests that MTC has a calorie 391 
restriction effect on C. elegans. 392 
 393 
3.6 Mobility is enhanced by MTC 394 
One interesting aspect of MTC research, is that it is thought to aid in muscle recovery 395 
after exercise (Bell, Stevenson, Davison, & Howatson, 2016; Howatson et al., 2010; 396 
Levers et al., 2015; Levers et al., 2016). Worms have only recently been shown to be 397 
suitable to use to investigate exercise and how this can be matched to humans 398 
(Laranjeiro, Harinath, Burke, Braeckman, & Driscoll, 2017). For this reason, we chose 399 
to test the ability of MTC to affect the burrowing capacity of C. elegans (Lesanpezeshki 400 
et al., 2019). Worms that are 1-week old have a striking reduction in their capacity to 401 
 17 
burrow through agar to reach a chemoattractant compared to L4 animals (Figure 6). 402 
However, exposure to just 16.7µl/ml of MTC from L4 for 1 week significantly enhanced 403 
the ability of animals to reach the surface of the gel (Figure 6). This was lost in animals 404 
exposed to higher doses of MTC. The pluronic gel burrowing assay could be 405 
considered an endurance challenge to the worms, who are more accustomed to 406 
moving on the surface of a plate. Strikingly, MTC has been shown in humans to aid in 407 
performance during endurance challenges (Levers et al., 2015; Levers et al., 2016), 408 
which is in sound conclusion with our data.  409 
 410 
4. Conclusion 411 
To date, there is promising evidence that MTC can contribute to the prevention of 412 
MetS development (Reis et al., 2016), although there is a lack of research examining 413 
the mechanism of action by which such effects are induced. Using C. elegans, our 414 
data provides encouraging evidence that MTC may be operating as a calorie 415 
restriction mimetic. Specifically, MTC is likely to act via the PPAR metabolic pathway, 416 
as has been shown previously in rats (Seymour et al., 2008) and is in sound 417 
agreement with other anthocyanins (Scazzocchio et al., 2011). Indeed, it is highly likely 418 
that the mechanism of action of PPAR activation is to operate as a calorie restriction 419 
mimetic (Corton et al., 2004) further supporting our findings.  420 
 421 
Additionally, our data supports the hormetic action of MTC. We have shown that worms 422 
exposed to MTC have an increase in fat content, and since lipids are highly susceptible 423 
to oxidative damage, the ability of worms to store more lipid indicates an increased 424 
resistance to oxidative stress. In recent years, lipid storage has been shown to be 425 
tightly coupled to suppression of oxidative stress and inflammation (van Heemst, 426 
 18 
2010). Reduced oxidative stress and inflammation after consuming MTC was shown 427 
in older human adults (Chai, Davis, Zhang, Zha, & Firschner, 2019; Johnson et al., 428 
2017; Traustadottir et al., 2009), providing translational evidence to the responses 429 
observed here in C. elegans. 430 
 431 
Together, this data highlights the use of C. elegans to search for nutritional 432 
interventions of health. Therefore, the result of this work is to provide a basis for further 433 
mammalian studies. Indeed, such data can then be used to design and perform more 434 
targeted longitudinal clinical trials in MetS humans. In addition, it is possible that genes 435 
identified in C. elegans as having a role in obesity (or diabetes) are likely to be 436 
implicated in human disease as well or, at the very least, provide candidates for anti-437 
obesity and anti-diabetic drugs.  438 
 439 
Acknowledgements 440 
We thank Michelle Severins, Chiara Pignato, Inge Vrinds and Ellen Obster for their 441 
support in the experiments at the HAN University.  442 
 443 
Funding: This work was supported by The University of Hertfordshire Diamond Fund 444 
and a Santander UK grant to TD, LB and MR. 445 
 446 
Author Contributions  447 
David van de Klashorst: Investigation, Methodology, Validation, Visualisation; 448 
Amber van den Elzen: Investigation, Visualisation; Jasper Weeteling: Investigation, 449 
Visualisation; Michael Roberts: Conceptualization, Formal Analysis, Funding 450 
Acquisition, Writing-reviewing and editing; Terun Desai: Conceptualization, 451 
 19 
Methodology, Formal Analysis, Funding Acquisition, Writing-reviewing and editing; 452 
Lindsay Bottoms: Conceptualization, Methodology, Formal Analysis, Funding 453 
Acquisition, Writing-reviewing and editing; Samantha Hughes: Conceptualization, 454 
Methodology, Formal Analysis, Resources, Visualisation, Supervision, Writing-455 
reviewing and editing 456 
 457 
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 709 
  710 
 30 
Table 1. The effect of silencing genes associated with fat metabolism in the 711 
presence of Montmorency tart cherry juice. Wild type animals were placed onto 712 
RNAi NGM seeded with, or without, 116.7µl/ml MTC, and after 48 hours at 20oC the 713 
developmental progress of the animals was compared. Control animals that are not 714 
exposed to MTC and are fed control RNAi (empty vector L4440) reach the L4/young 715 
adult stage (as shown by a green highlighted box and “normal development to L4/YA”). 716 
In contrast, MTC exposed worms have a significant developmental delay as indicated 717 
by a red box stating “enhanced developmental delay”. A selection of genes concerning 718 
fat metabolism are silenced in the presence of MTC and the development of the worms 719 
compared to the control was investigated. The silencing of most genes in the presence 720 
of MTC have no effect on developmental rate, with others show a recovery of the 721 
developmental delay (highlighted in green) and some further enhance the 722 
developmental delay (highlighted in red).  723 
  724 
 31 
 725 
Worm gene 
name 
Development 
after 48hours RNAi Human gene/description 
L4440 Normal development to L4/YA Empty vector control 
L4440 + MTC Developmental delay Empty vector control + MTC 
   
acl-12 Developmental delay 
Ortholog of LPGAT1; Acyl-
CoA:lysophosphatidylglycerol acyltransferase 
1 
alh-11 Developmental delay 4-trimethylaminobutyraldehyde dehydrogenase 
C17C3.3 Enhanced developmental delay Acyl-coenzyme A thioesterase 8 
C37H5.13 Developmental delay Acyl-coenzyme A thioesterase 8 
cyp-13A11 Developmental delay A nematode cytochrome P450 
daf-22 Normal development to L4/YA Isoform SCPx of Non-specific lipid-transfer protein 
daf-7 Developmental delay Gene involved in fat metabolism 
eat-2 Enhanced developmental delay Neuronal acetylcholine receptor subunit alpha-7 
elo-4 Normal development to L4/YA Polyunsaturated fatty acid elongase for very long chain fatty acids 
F09C8.1 Enhanced developmental delay Phospholipase B1, isoform 5, membrane-associated 
F25E2.3 Developmental delay Acyl-coenzyme A thioesterase 8 
fard-1 Developmental delay Fatty acyl-CoA reductase 1 
fat-3 Enhanced developmental delay fatty acid desaturase 1 
fat-4 Developmental delay fatty acid desaturase 1 
H27A22.1 Normal development to L4/YA Glutaminyl-peptide cyclotransferase-like protein 
klf-1 Enhanced developmental delay highly similar to Krueppel-like factor 5 
lipl-1 Enhanced developmental delay gastric triacylglycerol lipase isoform 1 
lipl-4 Normal development to L4/YA Lipase member M precursor 
M79.2 Developmental delay Glycerol-3-phosphate acyltransferase 3 
nhr-49 Enhanced developmental delay Isoform 1 of Hepatocyte nuclear factor 4-gamma 
nhr-80 Developmental delay Isoform HNF4-Alpha-2 of Hepatocyte nuclear factor 4-alpha 
oga-1 Developmental delay 
O-linked N-acetylglucosamine (O-GlcNAc)-
selective N-acetyl-beta-D-glucosaminidase 
(O-GlcNAcase) 
sbp-1 Normal development to L4/YA SREBP 
T19D7.7 Developmental delay Phospholipase B1, isoform 5, membrane-associated 
tub-1 Normal development to L4/YA Isoform 1 of Tubby protein homolog 
 726 
 727 
Table 2. Statistical analysis of lifespan. Raw lifespan data was analysed using the 728 
OASIS software (Yang et al., 2011) which provides the mean lifespan with standard 729 
error and 95% confidence interval (C.I.). For lifespan plots, see Figure 2. The control 730 
animals (no MTC exposure), lived for a maximum of 17 days, which was extended in 731 
 32 
all MTC exposed worms.  Of the animals exposed to 16.7µl/ml MTC, 50% mortality 732 
was extended from 10 to 13 days, and was the only dilution of MTC to show this. For 733 
each condition, over 100 animals were used, bar 150µl/ml where 80 animals were 734 
used. Intriguingly, the mean lifespan was only increased in animals exposed to the 735 
lowest dose of MTC, 16.7µl/ml. The Bonferroni correction was applied to show where 736 
the changes to lifespan were most significant. 737 
 738 
MTC 
concentration 
Number 
of 
worms 
Age in days at % 
mortality Restricted mean Bonferroni 
p value 50% 100% Days Std. Error 95% C.I. 
0µl/ml 112 10 17 11 0.31 10.07~11.27 - 
16.7µl/ml 111 13 23 12 0.43 11.44~13.12 0.0001 
66.7µl/ml 137 9 20 10 0.32 8.96~10.22 0.2358 
150µl/ml 81 8 20 9 0.35 7.90~9.29 0.0001 
 739 
Figure 1. MTC exposure causes a developmental delay at elevated 740 
concentrations. (A) Synchronised worms were placed on NGM (time = 0 hours) and 741 
observed daily. Worms were assayed for their development at 72 (i) and 86 (ii) hours 742 
post L1. Worms were assessed as being at a stage younger than L4 (white bars), L4 743 
(light grey bars) or egg laying adults (dark grey bars). Worms which died were recorded 744 
(black bars). Worms that were exposed to MTC concentrations above 100µl/ml died 745 
within 90 hours post development and are not shown. (B) A lawn clearance assay was 746 
undertaken as a proxy for brood size. L4 animals were added to NGM (time = day 0) 747 
and observed daily . The day on which the lawn was cleared was recorded.  Clearance 748 
on day 4 are the black bars, clearance after 5 days are very dark grey bars, while grey 749 
bars indicate a cleared lawn after 7 days. Light grey bars show where the lawn is 750 
cleared after 8 days. Where the bars are white, shows where it took 9 days or more to 751 
clear the lawn. No lawns were cleared on day 6. (C) Representative images of worms 752 
 33 
at different MTC concentrations after 72 hours growth on MTC from L1 stage. (i) 753 
Control animals are gravid. (ii) Animals exposed to 16.7µl/ml MTC have a similar 754 
development to control worms. (iii) After 66.7µl/ml and (iv) 88.7µl/ml MTC exposure, 755 
animals have a small developmental delay. (v) Worms exposed to 116.7µl/ml have a 756 
significant developmental delay. This animal is not dead, but has not yet reached the 757 
L4 stage and is clearly smaller than the other animals. Scale bar, 200µm. 758 
 759 
Figure 2. Lifespan of worms was extended following exposure to MTC. Wild type 760 
worms lived for a maximum of 17 days post L4 (solid black lines). Animals exposed to 761 
16.7µl/ml displayed an extension to lifespan throughout (grey dotted line). In contrast, 762 
animals exposed to 66.7µl/ml (dashed grey line) and 150µl/ml (solid grey line) showed 763 
a faster death rate compared to the control animals, but did have a slight extension to 764 
maximum lifespan. For each condition, over 100 animals were used, with the exception 765 
of 150µl/ml MTC where 80 animals were used. For statistical analysis, see Table 2.  766 
A
i. 72 hours post L1 ii. 86 hours post L1
Control
66.6µl/ml MTC
100µl/ml MTC
0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100
Percentage of animals at each stage Percentage of animals at each stage
0
10
20
30
40
50
60
70
80
90
100
Pe
rc
en
ta
ge
 o
f l
aw
ns
 c
le
ar
ed
Control 66.7µl/ml MTC 100µl/ml MTC
B
C
i. Control ii. 16.7µl/ml MTC iii. 66.7µl/ml MTC iv. 88.7µl/ml MTC v. 116µl/ml MTC
 34 
 767 
 768 
Figure 3. DAF-16 localisation is not affected by MTC. DAF-16 translocates from the 769 
cytoplasm to the nucleus to activate genes concerned with stress resistance and 770 
insulin signalling. Localisation of DAF-16::GFP (strain, TJ356) was scored as cytosolic 771 
(light grey), intermediate (dark grey) or nuclear (black). Under control conditions, DAF-772 
16::GFP is cytoplasmic however, there is a small shift in localisation when exposed to 773 
MTC. MTC exposure results in 7-15% animals having intermediate DAF-16::GFP and 774 
3% nuclear localisation. n=100 worms. 775 
 776 
0.25
0.50
0.75
1.00
0
0 5 10 15 20 25
S
ur
vi
va
l p
ro
ba
bi
lit
y
Time, days post L4
0
100
80
60
40
20
Control 66.7µl/ml MTC 100µl/ml MTC
P
er
ce
nt
ag
e 
of
 w
or
m
s
16.7µl/ml MTC
 35 
Figure 4. MTC exposed C. elegans show an increase in fat content. (A) 777 
Representative images of worms exposed to MTC. In all cases, animals were grown 778 
from L1 to L4 on NGM supplemented with increasing dilutions of MTC and the vital dye 779 
Nile Red. At L4 stage, worms were mounted for microscopy and imaged using identical 780 
settings. Images were taken at the anterior of the animal, with the focal plane always 781 
on the valve of the grinder. (i) Control animals, (ii) 16.7µl/ml MTC, (iii) 66.7µl/ml MTC 782 
and (iv) 83.3µl/ml MTC. Anterior is to the left, ventral to the bottom. Scale bar is 500µm.  783 
(B) Graph to show the relative fat staining in worms exposed to MTC. Images of worms 784 
exposed to MTC were analysed using ImageJ and the values of Nile Red staining are 785 
normalized to the control, and this value plotted. Control animals (black bars) show 786 
some Nile Red staining of the upper intestine. There is a significant increase in fat 787 
staining in the animals exposed to 16.7µl/ml and 66.7µl/ml MTC (grey bars) compared 788 
to control worms (black bar), while those worms exposed to 83.3µl/ml MTC (white bar) 789 
have a significant reduction in fat staining. Bars are averages and error is standard 790 
deviation. Statistical analysis utilized the t-test where ** is p<0.01 and n=26.  791 
 36 
 792 
Figure 5. Analysis of pharyngeal pumping activity when animals are grown in 793 
the presence of MTC. In all cases, L4 animals were screened in 2 independent 794 
experiments. Wild type animals are used in all cases. Control conditions are shown in 795 
black bars (n=35), 16.6µl/ml MTC are shown in grey (n=44) and 66.7µl/ml MTC are 796 
shown in white bars (n=43). Data is recorded as the average ± S.E.M, with all values 797 
being significantly different (p<0.001) unless otherwise stated. (A) Pump frequency, in 798 
Hz, is reduced in a dose dependent manner in worms that have been exposed to MTC. 799 
(B) Interpump interval, msec, issignificantly increased in worms that are exposed to 800 
MTC concentrations. There is no difference in IPI in worms exposed to MTC. (C) Pump 801 
duration, in msec, is significantly increased in MTC exposed worms compared to 802 
 37 
control conditions.  There is no difference in pump duration between MTC exposed 803 
worms. (D) Amplitude in µV is significantly increased in animals exposed to MTC, in a 804 
dose dependent manner.  805 
 806 
Figure 6. MTC enhances neuromuscular behaviour at low doses.  Animals will 807 
move from the bottom of the gel to the top, where there is a chemoattractant, and food. 808 
The majority of young, L4, animals (black line, circles) will reach the top of the gel 809 
within 90 minutes. However, older animals have muscle degeneration which can be 810 
viewed by fewer worms reaching the surface of the gel. Only half of the 1-week post 811 
L4 adults (dashed black line, circles) reach the surface of the gel after 2 hours. 812 
Interestingly, animals which have been exposed to 16.7µl/ml MTC (light grey, triangles) 813 
for 1 week have a similar rate of burrowing to the surface as control worms. In contrast, 814 
1-week old animals from 66.7µl/ml MTC (dark grey, diamonds) are worse than the 815 
 38 
similarly ages control worms, with just 40% animals reaching the surface of the gel. 816 
n=180 over 2 independent experiments.  817 
 818 
Supplemental Figure 1 RNAi knockdown affects development of animals 819 
exposed to MTC. Wild type animals were placed onto RNAi NGM seeded with, or 820 
without, 116.7µl/ml MTC, and after 48 hours at 20oC the developmental progress of 821 
the animals was compared. (A) Control animals that are not exposed to MTC and are 822 
fed control RNAi (empty vector L4440) reach the L4 stage (top panel). In contrast MTC 823 
exposed worms lag 1 day behind the non-exposed animals (bottom panel). (B) 824 
Representative images of worms, which when genes of interest were silenced in the 825 
presence of MTC showed a recovery of the developmental delay. Shown are 826 
H27A22.1, sbp-1, lipl-4 and daf-22. (C) Representative images of animals where RNAi 827 
silencing of genes enhanced the developmental delay. Two genes are shown, lipl-1 828 
and nhr-49. Scale bar is 500µm in all images. A list of the genes screened, and the 829 
results are shown in Table 1. 830 
 39 
 831