Wilcoxon test was used for comparing each treatment to its control in the real-time PCR analyses

All tests were performed using StataSE v12 software.Mushrooms are widely consumed foods whose high contents of bioactive compounds may provide antioxidant, anti-inflammatory, anti-obesity, and antidiabetic properties, among others.Thus, different in vitro and in vivo models have been used to identify and characterized the health benefits of these mushrooms for their potential application as food ingredients. Thereby, Grifola frondosa polysaccharides have been found to exert antioxidant and antidiabetic properties in different animal models . The regulatory pathways of energy homeostasis are highly conserved between C. elegans and mammals, making this nematode a powerful model for exploring the genetic bases of fatty acid synthesis and the regulation of fat storage. Thus, C. elegans has been widely used as a screening model for the identification and evaluation of BACs with healthy properties in the prevention of obesity-related disturbances, together with the characterization of the biological mechanism underlying these effects.

Moreover, thisnematode has been widely used for determining the antioxidant, anti-aging, and life-prolonging properties of BACs present in different food ingredients with beneficial properties in the prevention of aging-related diseases . Previous studies have suggested the anti-obesity properties of G. frondosa. For example, Aoki and colleagues demonstrated that supplementation with 0.4% G. frondosa extract in high fat-induced obese mice for 15 weeks significantly reduced body weight gain and visceral fat accumulation, ameliorated hepatic triglyceride storage, and improved glucose tolerance . They suggested that the anti-obesity and antidiabetic properties of this G. frondosa extract were attributed to its activity as a PPARδ agonist. As mentioned above, our GE represents an important source of different BACs, including beta-glucans, phenolic compounds, PUFAs, and MUFAs. Different studies have reported the lipid-reducing activity of different flavonoids and phenolic acids in C. elegans, including ours . For this reason, we aimed to determine if the combination of the bioactive compounds found in our GE could also affect the lipid homeostasis using the C. elegans model. The intestinal and hypodermal cells of this nematode accumulates lipids in the form of fat droplets, which can be detectable under microscopy using fat-soluble dyes, such as Sudan Black B, Oil red O, and Nile Red .

The quantification of the fluorescence of the fixative Nile Red lipophilic dye has been demonstrated to represent a reliable method to determine the fat content of this nematode, and has been widely used for evaluating the lipid-reducing activity of BACs, with potential uses in the prevention of and therapy for obesity-related diseases . For this experiment, L1 N2 wild-type worms were treated until reaching the L4 stage with and without the GE at the doses of 10 and 20 μg/mL, when nematodes were collected, fixed, and stained with Nile Red . As revealed by the quantification of the fluorescence of the worms , both doses of GE induced a significant reduction in the lipid content of C. elegans, in comparison with untreated control worms . Orlistat-treated worms were used as a positive control of fat reduction. In fact, the reduction induced by the high dose of GE was 18.64%, and a similar result was obtained after Oil Red O staining , confirming this effect on worm fat deposition. Although our extract did not exhibit in vitro genotoxicity in the SOS/umu test, these results could be related to an effect on nematode development. Thus, in order to dismiss this negative effect, we analysed the effect of GE extract treatment from L1 to day 1 of adulthood on worm length, size, and egg laying. No differences were observed in terms of worm length and size between GE-treated and untreated nematodes, suggesting that the lipid-reducing activity of GE is not accompanied by an effect on worm length and size. Furthermore, after 72 h of treatment, both GE and control plates exhibited the presence of both eggs and L1 larvae without differences in the time of appearance. All these results suggest that treatment with our GE from L1 to L4 significantly reduces the C. elegans fat content independently of any effect on worm development. Importantly, the effect of GE on fatty acid synthesis and breakdown was also accompanied by a tendency to higher expression of daf-2 , an ortholog of human IGF1R , INSR , and INSRR . This gene codes for the single receptor protein in the IIS pathway. Moreover, treatment with GE induced a pronounced overexpression of daf-16, the ortholog of human FOXO, which codes for a key transcription factor regulated by the IIS pathway. Daf-16 acts as a nutrient-sensing regulator of energy homeostasis and lipid metabolism .

The significant overexpression of GE on daf-16/foxo was also confirmed at a lower dose of the extract and would suggest that the anti-obesity properties previously observed with GE are mediated by the upregulation of this transcription factor. Finally, GE-treated worms exhibited a significant upregulation of skn-1 , an ortholog of the human NRF2 gene, an important transcription factor of the antioxidant and antiaging responses . No differences were observed in the expression of sod-3. Again, significant upregulation of skn-1 was observed after treatment with a lower dose of GE . SKN-1 activation has been previously shown to be involved in fat metabolism by depleting somatic lipids , so overexpression of this transcription factor by GE might also be involved in the fat-reducing activity observed in our Nile Red and Oil Red O analyses. Our findings demonstrate that our GE extract reduced the C. elegans lipid content when treated from L1 to L4; this effect is mediated by a reduction in the fatty acid biosynthesis and increased oxidation, together with a significant overexpression of the skn-1 and daf-16 transcription factors. In order to further investigate the potential implication of the daf-2/daf-16 and the skn-1/nrf-2 signalling pathways in the anti-obesity properties of the GE extract, we analysed the lipid-reducing activity of GE on C. elegans in a glucose-loaded medium.

Glucose has been used to establish a C. elegans obesity model in various studies , and has been demonstrated to affect both lipid accumulation and oxidative stress responses . Again, we observed that treatment with 20 μg/mL of GE induced a significant reduction in the fat content in comparison with untreated control worms in a glucoseloaded medium . A gene expression analysis performed after this assay demonstrated that, when the medium was supplemented with glucose, no differences were observed in skn-1 gene expression , suggesting that the skn-1 activation previously observed might be involved in the potential antioxidant activity of this extract, more than modulating the lipid accumulation in this model.