Xanthigen® reduces lipid deposition and improves stress resistance in Caenorhabditis elegans

#Both authors contributed and directed equally to this work. *Correspondence to Author: Laura López-Ríos Nektium Pharma SL, Las Palmas, Spain How to cite this article: María Dolores Jimenez, Laura López-Ríos, Rubén Pérez-Machín, Ricardo Chirino, Bárbara Davis, Elena Talero, Virginia Motilva. Xanthigen® reduces lipid deposition and improves stress resistance in Caenorhabditis elegans. Journal of Herbal Medicine Research, 2020,5:41. eSciPub LLC, Houston, TX USA. Website: https://escipub.com/ María Dolores Jimenez et al., JHMR, 2020, 5:41 Journal of Herbal Medicine Research (ISSN: 2474-106X) Research Article JHMR 2020, 5:41 Xanthigen® reduces lipid deposition and improves stress resistance in Caenorhabditis elegans Xanthigen® is a nutraceutical combination of two well-known natural products, brown seaweed extract (rich in fucoxanthin) and pomegranate seed oil (rich in punicic acid), and it has been designed to use in weight management, in conjunction with a calorie restricted diet. In the nematode Caenorhabditis elegans Xanthigen® treatment caused a significant reduction in lipid deposition in wild-type N2 (WT-N2) animals but not in sirt-2.1-de- ficient strain, which raises the possibility that the prolipolytic or anti-lipogenic effect of Xanthigen® in these animals is mediated through Sirtuin 2.1 activation. This response has been well described for Xanthigen® in cell cultures and other animal models. In addition, Xanthigen® treatment conferred to both strains an increased resistance to thermal and oxidative stress, which opens the possibility that the effects of Xanthigen® are not mediated solely by Sirtuin 2.1 activation. We therefore explored whether Xanthigen® could activate diverse defence mechanisms such as DAF-16 activation, or GST induction in response to xenobiotics, by using the strains TJ356, CL2070 and CL2166, stably expressing Pdaf-16::GFP, Phsp-16.2::GFP and Pgst-4::GFP, respectively. Xanthigen® treatment provoked neither DAF-16 translocation to the nucleus nor increased expression of HSP16.2 and GST4, which opens the possibility that different mechanisms other than DAF-16 and those involved in xenobiotic responses, are activated by Xanthigen® and are capable of conferring to the nematode an increased resistance to thermal or oxidative stress. Keywords: Xanthigen®, fucoxanthin, punicic acid, Caenorhabditis elegans, sirtuin 2.1, lipid deposition, thermal stress, oxidative stress María Dolores Jimenez1, Laura López-Ríos2*, Rubén Pérez-Machín2, Ricardo Chirino3, Bárbara Davis4, Elena Talero1#, Virginia Motilva1# 1Department of Pharmacology, Faculty of Pharmacy, Universidad de Sevilla, Seville, Spain; 2Nektium Pharma SL, Las Palmas, Spain; 3Department of Biochemistry, Molecular Biology and Physiology, Faculty of Health Science, University of Las Palmas de Gran Canaria (ULPGC), Las Palmas, Spain; 4P.L. Thomas & Co., Inc., NJ 07960, USA ABSTRACT JHMR: https://escipub.com/journal-of-herbal-medicine-research/ 1 María Dolores Jimenez et al. , JHMR, 2020, 5:41 JHMR: https://escipub.com/journal-of-herbal-medicine-research/ 2 INTRODUCTION Epidemiological studies have shown that overweight and obesity lead to serious health problems such as cardiovascular disease, diabetes, musculoskeletal disorders, and even cancer 1,2. In 2015, it was estimated that around 107 million children and 603 million adults were obese worldwide, with the prevalence being higher in women than in men of all ages3. Because overweight and obesity are the result of a sustained imbalance between caloric intake and caloric expenditure, lifestyle changes including calorie restriction and increasing energy expenditure are the two cornerstones in their treatment. Additionally, there are commercial formulations in the market that claimed as capable of enhancing the effects of lifestyle changes with a variable degree of scientific evidence 4, 5 . Xanthigen® is a proprietary patented combination of fucoxanthin, a carotenoid from brown seaweed extract (Undaria pinnatifida), and punicic acid, a conjugated fatty acid found in pomegranate (Punica granatum) seed oil, which helps to the weight management without toxic effects6. The effects of Xanthigen® are explained by the combination of properties of these two components. Many studies have shown anti-oxidant, anti-inflammatory, and anticancer activities of fucoxanthin8,9. In addition, it has been shown that fucoxanthin stimulated lipolysis and inhibited lipogenesis by increasing Uncoupling Protein 1 (UCP1) expression in both white and brown adipose tissue10. Moreover, in a clinical trial, this carotenoid has been shown to provoke a significant HbA1c (Glycated haemoglobin) reduction, in carriers of the G/G allele of the UCP1 gene (-3826A/G, rs1800592), suggesting its utility in genetically obese highrisk populations11. Punicic acid is a conjugated linolenic acid with antidiabetic, hypolipidemic and anti-inflammatory properties12. The combination of both compounds results in a more pronounced effect than that observed with fucoxanthin or punicic acid alone13,14. In clinical trials performed on non-diabetic overweight and obese women, Xanthigen® promoted weight loss, reduced body and liver fat content and improved liver function tests. In addition, Xanthigen® treatment improved blood lipid profile, inflammatory markers, blood pressure, and waist circumference, suggesting a potential role in the metabolic syndrome6. In a more recent interventional study performed on two healthy premenopausal obese women, Xanthigen® consumption induced the proliferation of Brown Adipose Tissue (BAT, which produces heat by non-shivering thermogenesis) in cervical, supraclavicular and paravertebral spaces in one of them, although body weight remained without changes15. The molecular action of Xanthigen® has been studied both in vitro and in vivo. Lai et al. (2012)16 reported that Xanthigen® suppressed accumulation of lipid droplets in differentiated 3T3-L1 adipocytes by modulating signaling pathways including Akt-dependent FoxOs signaling, as well as induction of Sirtuin-I (SIRT1). Similarly, Choi et al (2014)17 reported that the anti-obesity activity of Xanthigen® in high-fat diet-fed mice was associated with a decreased expression of PPARɣ, and 3- hydroxy-3-methylglutaryl-coenzyme A (HMG- CoA) reductase, and an increased activation of AMP-activated protein kinase (AMPK) α and β, and Acetyl-CoA carboxylase (ACC), with a reduction of leptin level and expression in adipose tissue and also the body weight. Sirtuins are NAD+-dependent protein deacetylases that are broadly conserved from bacteria to humans, and are involved in longevity and stress resistance. In the nematode Caenorhabditis elegans (C. elegans) these effects are mediated by the forkhead transcription factor DAF-16 (ortholog of the FOXO family of transcription factors encoded by a downstream gene in the insulin-like pathway). In fact, the effect of DAF-16 on life span extension in worms is dependent on a low insulin-like signalling and sir-2.1 overexpression18. Since Xanthigen® is capable of both reducing María Dolores Jimenez et al. , JHMR, 2020, 5:41 JHMR: https://escipub.com/journal-of-herbal-medicine-research/ 3 body fat and modulating gene expression involved in processes related to lipid storage, aging, and stress resistance (SIR-2.1, FoxOs, AMPk), we considered of interest to study whether this compound was able to confer resistance to stress in the nematode C. elegans, which is of utility as a model to report the influence of plant extracts on the process of ageing or stress resistance due to its short life span and the feasibility of manipulating cell signalling pathways by simple biotechnological methods19. MATERIALS AND METHODS Test Materials The wild-type C. elegans strain N2 (variety Bristol) (WT-N2) and the sirt-2.1-deficient strain VC199 sir-2.1 (ok434) IV were obtained from the Caenorhabditis Genetics Center (University of Minnesota, Minneapolis, USA), The C. elegans transgenic strains TJ356, zls356 (Pdaf-16: GFP); CL2070 drIs70 (Phsp-16.2: GFP); CL2166 davIs 19 (Pgst-4: GFP) were kindly donated by the laboratory of A. Miranda (Biomedical Institute of Seville, IBIS, Spain), and were maintained on 6 or 10 cm with nematode growth medium (NGM). NGM plates seeded with OP50 bacteria, according to standard methods20. All worms were cultured at 20°C. The strains were routinely propagated on NGM with E. coli strain OP50 as food and maintained at 20 °C. A volume of 100 µL of OP50 (overnight culture) was dropped on the midpoint of 60-mm NGM plates, which were allowed to dry overnight before worms were transferred. All experiments were repeated at least three times Xanthigen®, a nutritional supplement composed of brown seaweed extract containing 0.42 % of fucoxanthin and pomegranate seed oil, having 35% of punicic acid, was provided by Nektium Pharma (Canary Islands, Spain). To perform all assays, 1 mg powder of the product was added to the surface of NGM plates containing E. coli OP50. Reagents used in this work were provided by Sigma Aldrich (Spain) and Panreac Quimica (Spain). Oil Red O Staining C. elegans is widely used as a model for studying conserved pathways for fat storage and metabolism; we used for stained fat the Oil-Red- O Staining method (ORO)21. Both wild-type (WT-N2) and sir-2.1-defficient worms were incubated with either 1 mg/plate of Xanthigen® or vehicle (control group). After 5 days, synchronized worms were washed two times with 1x PBS pH 7.4 and allowed to settle by gravity, and resuspended in 120 µl of PBS to which an equal volume of 2x MRWB buffer (160 mM KCl, 40 mM NaCl, 14 mM Na2 EGTA, 1 mM Spermidine HCL, 0.4 mM Spermine, 30 mM NaPIPES, 0.2% βmercaptoethanol) containing 2% paraformaldehyde (PFA). Worms were then incubated one hour at room temperature; buffer was aspirated, and were washed with 1x PBS to remove PFA and were the resuspended in 60% isopropanol and incubated for 15 minutes at room temperature to dehydrate. Oil-Red-O stain solution 0.2% w/v in isopropanol was added, and animals were incubated overnight with rocking. The nematodes were washed tree times with 1x PBS 0.01% TritonX-100 and observed with an Olympus MVX10 microscope. Pixel 174 measurement from ORO stained worms was performed with Image J software. Stress condition evaluation All assays were carried out at 20 °C. Synchronized populations were obtained from larval stage (L1) to adults (L4 stage) in 60mm dishes. Plates with E. coli OP50, which serves as nematode food, were pre-treated with doses of 1 mg of Xanthigen® and dispersed as a thin layer. A control group exposed to the same solvent, dimethyl sulfoxide (DMSO) and Tween- 80 at 2% was used in parallel. Worms were judged to be dead when they ceased pharyngeal pumping and did not respond to prodding with a platinum wire. Sensitivity to oxidative stress was measured by quantifying survival following exposure to a reactive oxygen species (ROS)-generating compound such as juglone22. Age-synchronized María Dolores Jimenez et al. , JHMR, 2020, 5:41 JHMR: https://escipub.com/journal-of-herbal-medicine-research/ 4 nematodes (WT-N2 and sir-2.1-defficient worms) were incubated with 1mg of Xanthigen® or vehicle for 3 days in liquid NGM at 20ºC. The oxidative stress was induced with 240 µM juglone (5-hidroxi-1,4-naftoquinone) and worms were maintained at room temperature. The oxidative stress was evaluated as percentage of survival of the worms, and evaluated at 1, 2, and 3-hour using touch-provoked movement and ceased pharyngeal pumping. For thermal stress evaluation, synchronized 1- day-adult WT-N2 and -sir-2.1-defficient worms, pre-treated with 1 mg of Xanthigen® or vehicle (control) since L1, were exposed to 36.5°C for 8 h, thermal stress conditions23. Since 20ºC is considered the standard temperature for growing C. elegans in laboratory, after the procedure the plates were returned to this temperature for 24 additional hours. The thermal stress was evaluated as percentage (%) of survival of the worms at 6-hour using touch-provoked movement and ceased pharyngeal pumping. Overexpression of Pdaf-16::GFP, Phsp- 16.2::GFP and Pgst-4::GFP After synchronization, nematodes of the transgenic strains TJ356, CL2070 and CL2166, stably expressing Pdaf-16::GFP (FOXO transcription factor DAF-16), Phsp-16.2::GFP (heat shock protein 16.2), and Pgst-4::GFP (glutathionine-S-transferase-4) respectively, were transferred to NGM agar plates containing the respective compound or solvent control for additional 24 h. Subsequently, around 40 L3 larvae of each group were placed on microscope slides coated with 3% agarose, anaesthetized with 10 mM sodium azide, and covered with coverslips. As a positive control both of DAF16 and Hsp16.2, transgenic worms were grown at 20ºC plated with E. coli OP50 and exposed at 37ºC on the 3rd day of adulthood were used. As a positive control of GST4, transgenic worms were exposed to acrylamide at dose of 500 mg/L on the 3rd day of adulthood24. Cellular localization of DAF-16, Hsp16.2 and GST4 was analysed by fluorescence microscopy on an Axio Observer D1 Fluorescence Microscope (Zeiss, Göttingen, Germany) using appropriate filters (ex. 472±30 nm, em. 520±35 nm). All experiments were done in triplicate. Statistical analysis Data are expressed as the mean ± standard deviation (SD) of the mean. Statistical significance of differences between groups was analysed by one-way analysis of variance (ANOVA) followed by the Bonferroni post hoc test to allow for multiple comparisons. Statistical significance was reported if P<0.05 was achieved. RESULTS Effect of Xanthigen® on lipid deposition in C. elegans To study whether Xanthigen® was able to reduce lipid deposition in C. elegans, both WT- N2 (WT) and sir-2.1-defficient worms (sir-2.1) were treated with Xanthigen® or vehicle for five days and the extension of lipid deposition was then measured by the ORO method. Results are shown in Figure 1. WT animals treated with vehicle served as controls and their lipid deposition was considered to have a relative value of 1.000 ± 0.03. Treatment with Xanthigen® to WT animals caused a significant 13% decrease (0.871 ± 0.02) in lipid deposition as compared to controls (Bonferroni test: P<0.001). Sir-2.1-defficient worms exhibited a modest and non-significant 2.4% (0.976 ± 0.02) decrease in lipid deposition when compared to WT control (Bonferroni test: P=0.447). In these animals, Xanthigen® treatment caused a nonsignificant 3% decrement, in lipid deposition compared with sir-2.1 Cont (0.946 ± 0.02; Bonferroni test: P= 0.447). These results indicate that Xanthigen® is capable of reducing lipid deposition through a mechanism mediated by SIRT-2.1, although the involvement of other possible mechanisms cannot be ruled out. C. elegans converts excess energy into triglycerides that are stored in hypodermic cells of the intestine distinct droplet-like structures (Figure 2). WT worms exposed to Xanthigen® María Dolores Jimenez et al. , JHMR, 2020, 5:41 JHMR: https://escipub.com/journal-of-herbal-medicine-research/ 5 presented a lower intensity of colour detected by ORO method, which corresponds to a poorer content of triglycerides. This effect was reduced in the mutated sir-2.1, with no significant differences between Xanthigen® treated or nontreated worms. Effect of Xanthigen® on Oxidative stress To study the protective role of Xanthigen® on oxidative stress, juglone test, at a concentration of 240 μM, was added to wild type and sir-2.1- defficient worms, either in the presence or absence of Xanthigen®. Survival rate was assessed at 1, 2, and 3 hours. As shown in Figure 3, in WT worms, survival rate decreased with increasing times of juglone exposure (survival rate after 1 hour: 83.4 ± 4.3%; after 2 hours: 64.0 ± 4.5%; after three hours: 41.7 ± 3.9%). The addition of Xanthigen® to the incubation media significantly increased survival rate of the worms at each of the three times of incubation tested: 93.3 ± 2.5% after 1 hour (112% increase; Bonferroni test: P=0.012); 75.7 ± 3.2% after 2 hours (118% increase; Bonferroni test: P=0.006); 56.1 ± 2.4% after three hours (135% increase; Bonferroni test: P<0.001). María Dolores Jimenez et al. , JHMR, 2020, 5:41 JHMR: https://escipub.com/journal-of-herbal-medicine-research/ 6 Sir-2.1-defficient worms had a decreased survival rate as compared to wild-type animals at the three periods of exposure to juglone tested: 60.4 ± 4.8% after 1 hour (72% of the wild-type control; Bonferroni test: P<0.001); 37.3 ± 5.2% after two hours (58% of the wild-type control; Bonferroni test: P<0.001); and 16.8 ± 4.7% after three hours (40% of the wild-type control; Bonferroni test: P<0.001). In these animals, treatment with Xanthigen® provoked a significant increase in the survival rate at the three times examined: 75.0 ± 3.2% after 1 hour (124% increase; Bonferroni test: P<0.001); 51.6 ± 3.5% after 2 hours (138% increase; Bonferroni test: P<0.001); and 32.4 ± 3.3% after 3 hours (193% increase; Bonferroni test: P<0.001). These results indicate that sir-2.1 activation is able to confer worm resistance to oxidative stress. The fact that Xanthigen® was capable of increasing the survival rate after oxidative stress in both wild type and sir-2.1-defficient worms suggests that the effect may be independent of sir-2.1 activation. María Dolores Jimenez et al. , JHMR, 2020, 5:41 JHMR: https://escipub.com/journal-of-herbal-medicine-research/ 7 Effect of Xanthigen® on survival rate of worms exposed to thermal stress To study whether Xanthigen® was able to confer protection against thermal stress, adult, hermaphrodite worms of the two varieties, WT and sir-2.1-deficient worms, were cultured in NGM and exposed to thermal stress. As shown in Figure 4, survival rate after thermal stress was about 50% in WT worms (47.2 ± 5.8%). Xanthigen® treatment increased survival rate by 50% (72.0 ± 7.1%; P<0.001). In sir-2.1-defficient worms, survival rate was only 29.2 ± 5.6%, significantly lower than that exhibited by wildtype animals (P<0.001); treatment with Xanthigen® caused a significant 42% increase in survival rate (41.6 ± 6.1%; P=0.014). In fact, survival rate in sir-2.1-defficient animals treated with Xanthigen® was not significantly different than that exhibited by WT treated with vehicle (Bonferroni test: P=0.789), although was significantly lower than that exhibited by WT-N2 animals treated with Xanthigen® (Bonferroni test: P<0.001). These results suggest that sir-2.1, upon activation after thermal stress participates in the mechanisms that confer protection against thermal damage. Since Xanthigen® is capable of protecting worms against thermal stress both in the presence or absence of SIR-2.1, it opens the possibility that Xanthigen® exerts its protective effect on the animals through mechanisms independent of sir-2.1 activation. María Dolores Jimenez et al. , JHMR, 2020, 5:41 JHMR: https://escipub.com/journal-of-herbal-medicine-research/ 8 Effect of Xanthigen® on DAF-16 translocation, HSP-16.2, and GST overexpression In order to evaluate other possible mechanisms implicated in the protective effect of Xanthigen® on stress resistance, we used specifics worm strains to study the expression of proteins with a well-known role in response to stress. DAF-16 plays a role in both oxidative and heat stress as well as starvation. It is the sole orthologous of the FOXO family of transcription factors in C. elegans and DAF-16/FOXO proteins are a family of transcription factors that integrate different signals by phosphorylation of proteins including insulin/IGF-1, TOR, AMPK, or JNK pathways to modulate important processes, via shuttling from cytoplasm to nucleus, and the expression of genes that influence in cell growth, proliferation, differentiation, or longevity25. Under these circumstances, cytosolic DAF-16 protein is phosphorylated and then it relocates to the nuclei and promotes both the expression of genes that confer resistance to stress and longevity. The product of one of these genes is HSP-16.2, which can be considered as a marker of DAF-16 activation. In the absence of thermal stress, DAF-16 is located in the cytoplasm and Hsp-16.2 expression is very low. Under heat shock DAF-16 is translocated to the nuclei and promotes Hsp16.2 expression. Worms were assigned to three groups: the negative control was neither treated with María Dolores Jimenez et al. , JHMR, 2020, 5:41 JHMR: https://escipub.com/journal-of-herbal-medicine-research/ 9 Xanthigen® nor exposed to thermal shock at 37 ºC for 1 hour; the positive control was exposed to thermal stress but did not receive Xanthigen, and finally, the third group did not undergo thermal stress but was treated with Xanthigen®.