Coffee Silverskin Supplementation Alleviates High-Glucose-Diet-Induced Obesity by Modulating Lipogenic Gene Expression in Caenorhabditis elegans Model

Abstract

Coffee silverskin (CSS), the major by-product of coffee roasting, is reported to contain bioactive compounds, including xanthines and polyphenols, showing promising potential for food and nutraceutical applications. This study investigated the beneficial effects of CSS hydroalcoholic extracts, which were chemically characterized by Attenuated Total Reflectance–Fourier-Transform Infrared Spectroscopy and ElectroSpray Ionization tandem Mass Spectrometry, on Caenorhabditis elegans physiology. CSS supplementation improved healthspan-related parameters and delayed aging-associated functional decline, without significantly extending lifespan in wild-type nematodes. Treated worms exhibited a 57% reduction in reactive oxygen species (ROS) levels and upregulation of antioxidant genes (gst-4 and sod-3), suggesting that CSS mitigates oxidative stress through the DAF-2/DAF-16 pathway. Under high-glucose diet conditions, CSS reduced lipid droplet accumulation and modulated the expression of metabolic genes, including upregulation of nhr-49 which is a key regulator of fatty acid oxidation. CSS restored lipid homeostasis and rescued the shortened lifespan of obese nhr-49 mutant worms, suggesting enhanced β-oxidation. Moreover, CSS modulated serotonergic signaling by increasing tph-1 and ser-6 expression, linking its effects to serotonin-mediated regulation of fat metabolism. Finally, CSS promoted the growth of probiotic strains, suggesting potential prebiotic properties. Overall, these findings identify CSS as a metabolic modulator capable of alleviating oxidative and metabolic stress, supporting its sustainable application in the development of functional foods and nutraceuticals.

1. Introduction

Coffee silverskin (CSS) is the only by-product of the coffee roasting phase, representing a consistent quantity of waste, since, for every eight tons of coffee roasted, approximately 60 kg of CSS are produced [1]. CSS is currently employed primarily as a direct fuel for composting and soil fertilization [2,3]. However, its chemical profile rich in bioactive compounds suggests potential use as a food ingredient [4].

In recent years, the concept of sustainability has become crucial in addressing the challenges faced by the global food system. One major challenge is food waste, which not only discards valuable resources but also significantly harms the environment. Within the farm-to-fork framework, innovative solutions are essential to mitigate this issue and enhance the efficiency of the food supply chain [5]. The Farm-to-Fork strategy emphasizes resilient food systems, promoting sustainable diets, consumer empowerment, and global sustainability standards. The first step to address food waste is prevention, as most environmental impacts occur during the production phase [6]. In addition to food waste prevention, food waste valorization is increasingly important as it offers a sustainable solution to mitigate these massive economic and environmental impacts. In total, 44–47% of food wasted annually consists of fruits, vegetables, meat, and fish. Recycling and converting food waste into valuable products can significantly contribute to achieving global sustainability goals and promoting environmentally sustainable development [7].

Attention is focused on the valorization of agri-food by-product waste (FW) that presents an opportunity to produce value-added products for different industries like cosmetics, pharmaceuticals, and food/nutraceuticals. These by-products are rich sources of bioactive compounds, including polyphenols, tannins, flavonoids such as anthocyanins, vitamins A and E, essential minerals, fatty acids, volatile compounds, and pigments, obtained using various extraction techniques [8]. These bioactive compounds not only enhance the nutritional value of products but also offer various health benefits such as gut microbiota modulation, immunostimulation, antioxidant and anti-inflammatory activity [9].

Non-edible parts of fruits such as peels or skin portions and twigs often contain higher amounts of bioactive compounds when compared to the edible parts [10]. For example, peels of apple, grapes, citrus fruits and seeds of jackfruit, avocado and mango are reported to have more than 15% higher contents of polyphenolic compounds than pulps [11]. To investigate the effect of these bioactive molecules, tests on simple models’ systems are needed. In this context, the nematode Caenorhabditis elegans emerges as a promising model system for high-throughput screening of natural bioactive substances and for exploring their potential such as the antioxidant properties, as well as their effects on anti-aging and anti-obesity mechanisms [12]. The characteristics that make C. elegans particularly suitable for studying the impact of bioactive compounds on various biological processes include its transparent body, short life cycle, and ease of handling. Additionally, its complete genome sequence being available since 1998, the wide availability of mutants, and the low costs associated with its maintenance make it an ideal model organism. Furthermore, its capability for both self-fertilization and sexual reproduction adds to its versatility in research applications. The antioxidant activity of bioactive compounds can be effectively evaluated in C. elegans because this model organism possesses conserved cellular pathways, including the insulin/insulin-like growth factor 1 (IIS) signaling pathway, the target of rapamycin (TOR) signaling pathway, and the AMP-activated protein kinase (AMPK) signaling pathway, involved in oxidative stress responses and aging [13,14]. In addition, the nematode is a valuable model for studying the anti-obesity potential of natural compounds because of its conserved energy metabolism and fat regulatory pathways.

While the antioxidant activity of CSS has been extensively characterized across various models, its potential impact on complex physiological processes that go beyond mere oxidative stress mitigation remains largely unexplored. The present study departs from this traditional focus to provide a systematic in vivo investigation, utilizing the C. elegans model system, into the role of CSS in the active regulation of lipid metabolism. Specifically, this work aims to elucidate novel mechanisms of action by focusing on the modulation of NHR-49 and serotonin signaling pathways, based on the hypothesis that CSS could alleviate obesity-related phenotypes induced by a High-Glucose Diet (HGD). To validate this hypothesis, an obesity model was induced by feeding C. elegans animals 2% glucose. We then observed the effects of CSS on physiology, analyzing lifespan, aging, oxidative stress response and lipid metabolism.

2. Results

2.1. Chemical Characterization of CSS by ATR-FTIR Spectroscopy

The infrared spectra of CSS powder and its hydroalcoholic extract (CSS extract) are presented in Figure 1. The spectrum of CSS powder (Figure 1, spectrum a) presents a broad band around 3300–3400 cm⁻¹ which corresponds to the stretching vibration of hydroxyl groups (–OH) from polysaccharides and lignin. The signals at 2920 and 2852 cm⁻¹ are attributed to the asymmetric and symmetric C–H stretching vibrations. In the region between 1700 and 1500 cm⁻¹ the signal at 1614 cm⁻¹ can be attributed to the C=C stretching of aromatic rings in lignin, while the shoulder at 1516 cm⁻¹ is indicative of aromatic skeletal vibrations of lignin [15,16]. In the FTIR spectral region between 1420 and 1200 cm⁻¹, the spectrum exhibits several bands indicative of various molecular vibrations. These include C–H deformation vibrations (scissoring and bending), C–O stretching, and ring vibrations associated mainly with polysaccharides and lignin [17]. The strong band in the range between 1200 and 1000 cm⁻¹, centred at 1028 cm⁻¹ corresponds to C–O and C–O–C stretching of polysaccharides, particularly cellulose and hemicellulose [18,19]. The presence of cellulose is also confirmed by the band at 895 cm⁻¹, typical of the β-glycosidic stretching of β-1,4 cellulose [20].

Figure 1. Infrared spectra of coffee silverskin (CSS powder, spectrum a) and its hydroalcoholic extract at the concentration of 250 µg/mL (CSS extract; spectrum b).

In the spectrum of CSS extract (Figure 1, spectrum b), the bands in the region of 3400–3200 cm⁻¹ indicates a symmetric and asymmetric stretching of O–H groups [21]. The signal at 3009 cm⁻¹ is attributed to C–H symmetric stretching of the cis double bonds (H–C=C–H) of unsaturated fatty acids [22]. Also, the signals at 2922 cm⁻¹ and 2852 cm⁻¹ correspond to asymmetric and symmetric C–H stretching vibrations of aliphatic chains of lipids [23]. A distinct absorption at 1736 cm⁻¹ can be attributed to C=O stretching vibrations of ester and carboxylic acid groups, possibly arising from lipid esters or chlorogenic acid derivatives [24,25]. Two distinct signals at 1658 cm⁻¹ and 1633 cm⁻¹ are attributed to aromatic C=C stretching and C=O stretching, consistent with aromatic structures typically associated with hydroxycinnamate-containing extracts [24]. The absorption at 1515 cm⁻¹ is also associated with aromatic skeletal vibrations of phenols [26]. The absorption centred at 1467 cm⁻¹ can be assigned to CH₂ bending vibration of lipids [22], while the signal at 1418 cm⁻¹ is characteristic of the O–H bending of phenolic hydroxyls [27]. The signal at 1134 cm⁻¹ is assigned to C–O stretching vibrations of esters [22] and the band at 965 cm⁻¹ can be ascribed to out-of-plane bending of alkenes (–HC=CH–), indicating the presence of unsaturated fatty acids [22]. The signal at 703 cm⁻¹ can therefore be assigned to both the aromatic C–H out-of-plane bending of phenols [22] or to rocking vibrations of CH₂ groups in lipids [22]. The signal at 635 cm⁻¹ is associated with out-of-plane ring bending of aromatic compounds, particularly phenols [26].

2.2. Compound Identification by Mass Spectrometry

Direct-infusion electrospray ionization tandem mass spectrometry (ESI-MS/MS) was employed to identify the main compounds present in the extract. The identified compounds are summarized in Table 1 and include phenolic compounds, with several derivatives of hydroxycinnamic acids, organic acids, and the alkaloids caffeine and trigonelline.

Table 1. ESI-MS/MS characterization of compounds in coffee silverskin hydroalcoholic extract.

Compound	Precursor Ion (m/z)	Product Ions (m/z)	Polarity	Identification
Hydroxycinnamic acids derivatives
Di-caffeoyl quinic acid	514.9	353, 179, 173	negative	standard
Feruloylquinic acid	367.5	193, 173, 134	negative	[28]
Chlorogenic acid	353.1	191, 179, 135	negative	standard
Coumaroylquinic acid	337.3	191, 173, 155	negative	[28]
Caffeic acid	179.8	161, 135, 89	negative	standard
Dihydroferulic acid	195.2	177, 136,	negative	standard
Coumaric acid	163.1	135, 119, 93	negative	standard
Other phenolic compounds
Gallic acid	169.3	151, 125	negative	standard
Pyrogallol	125	107, 97, 81, 79	negative	standard
Alkaloids
Caffeine	195.0	138, 83, 63	positive	standard
Trigonelline	138	110, 94	positive	[29]
Organic acids
Quinic acid	191.2	127, 111, 93, 85	negative	standard
2-Furoic acid	111.3	83, 67	negative	[30]

2.3. Evaluation of Prebiotic Potential and of Antimicrobial Activity

The study of the potential prebiotic activity of CSS, chemically characterized in [4], was conducted through the evaluation of the effect exerted on the growth of probiotic bacteria, adapting the experimental procedure published by [31]. Briefly, the procedure is based on the replacement of the carbon source normally used in bacterial culture medium with the compound to be tested, and on the comparative evaluation of bacterial growth on the different carbon sources. This experimental system, although representing a simplified context as compared to in vivo or fecal fermentation models, is fast and economical, allowing to obtain preliminary information useful for selecting promising prebiotic candidates to be subjected to more in-depth analyses.

The experiments were conducted using commercial probiotics Lacticaseibacillus rhamnosus GG^®, Limosilactobacillus reuteri DSM 17938^®, Lacticaseibacillus paracasei CNCMI-1572^®, and Bifidobacterium longum W11^®. Each probiotic strain, previously grown in complete medium to stationary phase, was inoculated at the initial concentration of 1 × 10⁶ cells/mL into the corresponding medium without carbon source (normally represented by glucose), used as a negative control, or in the same medium added with glucose (conventional carbon source), inulin (control prebiotic), or CSS. After 24 h of incubation, bacterial growth was monitored by measuring log CFU/mL, comparing the values obtained for the CSS-containing medium with those obtained for the different control media. The results obtained, reported in Figure 2, show that all the four probiotic strains were able to grow using CSS as a carbon source, reaching log CFU/mL values ranging from 7.2 to 9. For Lacticaseibacillus rhamnosus GG^® and Lacticaseibacillus paracasei CNCMI-1572^®, total viable counts in the presence of CSS were similar to those obtained in the presence of glucose or inulin. Growth in the absence of carbon source (blue bars) was negligible, therefore the potential growth support from other medium components present in MRSnoGlu was excluded. Concerning the antimicrobial activity, no effect was detected against the tested pathogens.

Figure 2. Total viable counts (expressed as log CFU/mL) of Lacticaseibacillus rhamnosus GG^®, Limosilactobacillus reuteri DSM 17938^®, Lacticaseibacillus paracasei CNCMI-1572^® and Bifidobacterium longum W11^®, after 24 h growth in MRS without glucose (blue bars) or added with glucose (orange bars), inulin (green bars), CSS (light blue bars). Bars represent the geometric mean ± SD of three independent experiments. Statistical analysis was performed by ANOVA with a post hoc Tukey honestly significant difference (HSD) test. Different letters indicate significant differences (p < 0.005).

2.4. CSS Supplementation Affects Body Lenght but Not Lifespan in C. elegans

To investigate the potential beneficial effects of CSS extract, an in vivo lifespan assay was conducted (Figure 3a, Table S2). In this experiment, nematodes were fed with a standard diet of heat-killed E. coli OP50 or supplemented with three different concentrations of hydro-alcoholic CSS extracts, 2.5 µg/mL, 25 µg/mL, and 250 µg/mL, starting from the embryo hatching. Generally, a wide range of CSS concentrations has been tested in toxicological studies, performed in vitro and in vivo, as reviewed by Lorbeer et al. 2022 [32]. The three concentrations used in the present work were selected based on noncytotoxic levels reported to investigate CSS effect on cell redox status ([32] and references therein). Figure 3a shows that none of the tested CSS concentrations significantly affected worm viability, with 50% survival observed on day 15 and 16 for nematodes supplemented with 250 µg/mL and 25 µg/mL CSS, respectively, compared to day 17 for the control group, indicating the absence of toxic effects.

Figure 3. (a) Kaplan–Meier survival plot of wild-type worms fed with heat-killed OP50 bacteria and supplemented with CSS at concentrations of 2.5, 25, and 250 µg/mL. For each data point, n = 80 for each condition per experiment (ns: not significant). The lifespan assay was performed in triplicate, using worms fed with OP50 as a reference (untreated). (b) Average number of embryos produced per worm in animals supplemented with different CSS concentrations. Bars represent the mean of three independent experiments (n = 10 for each condition). Statistical analysis was performed using one-way ANOVA followed by the Bonferroni post-test; asterisks denote significant differences (ns: not significant). (c) Effect of CSS supplementation on the body size of C. elegans. Nematode length was measured from head to tail at the indicated time points. Bars represent the mean of three independent experiments (n = 10 for each condition), with asterisks indicating p-values (log-rank test) normalized to the untreated control (*** p < 0.001; ns: not significant).

To evaluate whether CSS may impact healthspan, a brood size analysis was conducted (Figure 3b). The results revealed no significant variation in the number of progeny produced across different treatment conditions. Additionally, body length was measured as a key parameter to evaluate the impact of CSS supplementation on the worms (Figure 3c). No significant differences in body length were observed between the control group and any of the three CSS concentrations (2.5 µg/mL, 25 µg/mL, and 250 µg/mL) on days 2 and 3 post-hatching. However, by day 4, worms treated with 25 µg/mL CSS exhibited a significant 10% increase in body length compared to the control. By day 7, worms treated with both 25 µg/mL and 250 µg/mL CSS showed a similar 10% increase in body length, indicating that CSS promotes growth in later developmental stages, although no clear dose-dependent effect was observed between the effective concentrations.

2.5. CSS Extract Enhances Healthspan in Aged Nematodes

To evaluate the effects of CSS extract on aging, age-related biomarkers were assessed. Pharyngeal pumping is a critical physiological function that reflects the health and vitality of nematodes. This process involves the contraction of the pharyngeal grinder, essential for feeding, and is known to decline with age, making it a valuable marker for assessing age-related changes [33]. As shown in Figure 4a, pumping analysis revealed that 10-day-old adult nematodes supplemented with 25 µg/mL and 250 µg/mL of the hydro-alcoholic extract exhibited a significant increase in pumping rate compared to the control group, with an enhancement of approximately 17% and 20%, respectively. In contrast, supplementation with 2.5 µg/mL did not result in a significant change compared to the control. Moreover, body locomotion measures the nematode’s ability to move and navigate its environment, which typically declines with age. As shown in Figure 4b, the body locomotion analysis revealed no significant differences in movement among 10-day-old nematodes treated with various concentrations of CSS extract compared to the control.

Figure 4. (a) Pumping rate of worms at 10 days of adulthood after treatment with various concentrations of CSS from embryo hatching, measured over a 30 s interval. Pharyngeal contractions were calculated as the average from 10 worms per condition. (b) Body locomotion ability measured over 30 s in 10 worms treated with different concentrations of CSS after 10 days of adulthood. Worms fed heat-killed OP50 without CSS supplementation were taken as controls (untreated). Statistical analysis was performed using one-way ANOVA followed by the Bonferroni post-test; asterisks denote significant differences (* p < 0.05, ** p < 0.01, ns: not significant). Bars represent the mean of three independent experiments.

2.6. CSS Supplementation Reduces ROS Accumulation Through IIS Pathway

Reactive oxygen species (ROS) are key drivers of oxidative stress and aging, leading to cellular damage [34]. Cells reduce ROS through detoxifying enzymes such as catalase and superoxide dismutase (SOD), which neutralize oxidative molecules. Studying the effects of agri-food byproducts like CSS on ROS levels and these detoxifying enzymes helps evaluate their potential to mitigate oxidative stress and promote health. As shown in Figure 5a, 1-day-old adult nematodes supplemented with 25 µg/mL of CSS, selected as the lowest effective dose showing consistent physiological effects, exhibited a 40% increase in ROS compared to the control. ROS levels were subsequently measured in 10-day-old adults, where the control group showed the expected age-related increase in ROS compared to 1-day-old controls. In contrast, a 57% reduction in ROS levels was observed in aged nematodes supplemented with CSS compared to the aged control group. Supplementation with 25 µg/mL CSS led to a 40% reduction in ROS levels in 10-day-old adults compared to their 1-day-old counterparts, indicating a protective effect of CSS against age-related oxidative stress. This reduction in ROS is supported by the upregulation of detoxifying enzymes, gst-4 and sod-3, in 1-day-old nematodes (Figure 5b), showing increases of over two-fold and 60%, respectively, compared to the control. This suggests that CSS enhances the cellular antioxidant response, contributing to the observed decline in ROS levels.

Figure 5. (a) Measurement of Reactive Oxygen Species (ROS) levels in N2 worms supplemented with 25 µg/mL CSS compared to untreated controls. Experiments were performed in triplicate (n = 50 for each condition), and data are presented as mean ± SD (** p < 0.01). (b) Expression of the detoxifying enzymes sod-3 and gst-4 genes in N2 worms treated with 25 µg/mL CSS or untreated control at day 1 of adulthood. Histograms display gene expression related to oxidative stress, as detected by real-time PCR (*** p <0.001, n = 200 for each condition).

In C. elegans, the DAF-2/DAF-16 pathway and the MAPK cascade (involving PMK-1, SEK-1, SKN-1, and HSF-1) are crucial for regulating oxidative stress and immune responses [14]. Under stress, reduced DAF-2 signaling activates the transcription factor DAF-16, which translocates to the nucleus and promotes the expression of stress-resistance genes, including antioxidant enzymes such as SOD and catalase [35]. Real-time PCR results showed that daf-2 expression was more than doubled in CSS-treated worms compared to controls, indicating activation of the DAF-2/DAF-16 pathway (Figure 6). Additionally, a 70% increase in daf-16 expression in 1-day-old adult worms supplemented with CSS compared to the control, aligning with the reduced ROS levels. Regarding the MAPK cascade, PMK-1 kinase and the transcriptional factor SKN-1, both involved in stress responses and immunity [36], showed no significant difference in expression between CSS-treated worms and controls, suggesting that CSS may not significantly affect this pathway. Similarly, SEK-1, the upstream MAP kinase kinase that activates PMK-1, was expressed at lower levels in CSS-treated worms, indicating reduced activation of the MAPK pathway (Figure 6). In contrast, HSF-1, a key regulator of heat shock proteins involved in stress protection [37], was significantly upregulated in CSS-supplemented worms, with expression levels 1.5 times higher than those of the control nematodes fed with heat-killed OP50. This suggests that CSS enhances the stress response by promoting heat shock protein production, which contributes to improved cellular resilience under stressful conditions and protects cells from oxidative damage.

Figure 6. Expression levels of daf-2, daf-16, pmk-1, sek-1, skn-1, and hsf-1 genes in 1-day-old adults treated with 25 µg/mL CSS or left untreated (control). Histograms display the transcript levels of the indicated genes, as measured by real-time PCR. Statistical analysis was performed using two-way ANOVA followed by the Bonferroni post-test. Experiments were performed in triplicate (n = 200), and data are presented as mean ± SD (* p< 0.05; ** p < 0.01; *** p < 0.001; ns: not significant).

2.7. CSS Reduces Lipid Droplets Accumulation in Obese Model

Testing CSS in C. elegans under High-Glucose Diet (HGD) conditions helps evaluate its potential to counteract oxidative stress and aging. An in vivo lifespan assay was conducted on NGM plates supplemented with 2% glucose to simulate obesity and using the three concentrations of CSS (Figure 7a, Table S2). The results revealed that worms treated with 25 µg/mL and 250 µg/mL CSS exhibited a 50% survival rate on day 8, like untreated controls. Overall, although the survival curves are superimposed for all the three concentrations, at the lowest concentration tested (2.5 µg/mL), a slight reduction was observed when the population reached 50% of survival, as compared to the control.

Figure 7. (a) Kaplan–Meier survival plot of HGD worms treated or not with 25 µg/mL CSS (control: untreated). n = 80 for each data point of single experiments (not significant). (b) Pumping rate of 10-day-old worms, measured over 30 s and determined from the average of 10 worms for each condition. (c) Body bending of C. elegans fed with CSS compared to untreated worms, measured over 30 s. Worms fed OP50 without CSS supplementation were used as controls. Statistical analysis was performed using one-way ANOVA followed by the Bonferroni post-test; asterisks indicate significant differences (* p < 0.05, *** p < 0.001, ns: not significant). Bars represent the mean of three independent experiments.

On the other hand, pharyngeal pumping rates showed a significant 10% increase across all CSS treatments compared to the control, indicating improved feeding function (Figure 7b). However, body bends analysis showed no significant differences among the CSS-treated groups and the control, suggesting that CSS did not noticeably impact locomotion (Figure 7c).

Lipid droplets (LDs) are indicators of lipid metabolism and storage, often increasing under conditions like obesity and metabolic stress [38]. The impact of CSS supplementation on lipid droplet accumulation was assessed in C. elegans under HGD conditions, simulating metabolic stress. Figure 8a shows a significant reduction in lipid droplet accumulation at the highest CSS concentration (250 µg/mL) compared to the HGD control and similar to worms grown in standard conditions. In contrast, the lower concentrations (25 µg/mL and 2.5 µg/mL) exhibited a progressive increase in lipid droplets, with levels nearing those seen in the HGD control population. These findings suggest that higher CSS concentrations effectively mitigate lipid accumulation, while lower concentrations do not offer the same protective effect, indicating a concentration-dependent response to CSS in managing lipid storage under metabolic stress. This result was further confirmed by the Mean Fluorescence Intensity (MFI) data (Figure 8b). The MFI, reflecting lipid droplet content, shows an inverse relationship with CSS concentration in HGD nematodes. As the CSS concentration decreases, MFI increases, approaching levels observed in HGD nematodes.

Figure 8. (a) BODIPY™ 493/503 staining of lipid droplets in 1-day-old adult worms fed OP50 and supplemented with different concentrations of CSS. Scale bar: 50 μm. (b) Median fluorescence intensity of worms stained with BODIPY. Statistical analysis was performed using one-way ANOVA followed by the Bonferroni post-test; asterisks denote significant differences (ns: not significant; *** p  <  0.001). Bars represent the mean of three independent experiments with n  =  30.

To provide a more comprehensive characterization of the lipid profile, we also performed Oil Red O (ORO) staining (Figure S1). Consistent with the BODIPY results, the ORO analysis confirmed a significant reduction in neutral lipid and triglyceride stores in CSS-treated nematodes, reinforcing the ability of the extract in modulating lipid accumulation under metabolic pressure.

2.8. CSS Treatment Counteracts Glucose Toxicity Through Alteration of Lipid Metabolism Pathways

To investigate the effects of CSS supplementation on lipid metabolism, we analyzed the transcript levels of key genes involved in fatty acid synthesis, desaturation, and energy homeostasis, including sams-1 (S-adenosylmethionine synthase), fat-7 (Delta-9 fatty acid desaturase), fasn-1 (Fatty Acid Synthase), sbp-1 (Sterol Regulatory Element Binding Protein), and pod-2 (Polarity and Osmotic Sensitivity Defect), which play crucial roles in lipid biosynthesis and metabolic regulation [33,39,40,41,42]. As shown in Figure 9a, High-Glucose Diet (HGD) conditions led to a significant upregulation of lipid synthesis genes compared to the control population grown under standard conditions. Notably, supplementation with 250 µg/mL CSS effectively counteracted this increase, restoring gene expression levels closer to the untreated control. In contrast, the lower CSS concentration (25 µg/mL) failed to mitigate the HGD-induced upregulation, suggesting that a higher dose is required to modulate lipid synthesis effectively. Figure 9b illustrates the transcript levels of genes associated with fatty acid β-oxidation (cpt-1, hacd, acs-2, tph-1, nhr-49, ser-6) and lipolysis (atgl-1). HGD conditions significantly reduced the expression of β-oxidation genes compared to standard diet control, indicating an impaired lipid breakdown capacity. However, supplementation with 250 µg/mL CSS restored β-oxidation gene expression to control levels or even higher, as observed for hacd, nhr-49, and tph-1. Interestingly, atgl-1 expression was approximately 30% lower following CSS treatment compared to both the untreated control and HGD conditions, suggesting a potential modulation of lipolytic activity (Figure 9b). A particularly intriguing finding was the effect of CSS on serotonin-mediated metabolic regulation. tph-1, encoding tryptophan 5-monooxygenase, the rate-limiting enzyme in serotonin biosynthesis, exhibited partial recovery following treatment with 250 µg/mL CSS, suggesting an adaptive response to metabolic stress. Downstream tph-1, the gene encoding for serotonin receptor ser-6, which enables octopamine receptor activity and plays a role in the positive regulation of fatty acid β-oxidation, was upregulated two-fold in 250 µg/mL CSS-treated worms compared to the HGD group, though it did not fully return to control levels (Figure 9). Notably, 25 µg/mL CSS supplementation led to a moderate 30% increase in ser-6 expression, indicating a dose-dependent response. These findings suggest that CSS modulates lipid metabolism via both direct transcriptional regulation of fatty acid oxidation genes and probably involving serotonin signaling, further supporting its role as a metabolic modulator under conditions of nutritional stress. Serotonin also plays a crucial role in regulating feeding behavior and metabolic homeostasis in C. elegans. To assess whether CSS supplementation influences serotoninergic signaling, we analyzed the expression levels of ser-4, which encodes a serotonin receptor involved in feeding regulation [43]. Real-time PCR analysis revealed a significant upregulation of ser-4 transcripts following treatment with both 250 µg/mL and 25 µg/mL CSS, with a 50% increase compared to the HGD condition, suggesting potential contribution of serotonin pathway to the observed increase in pharyngeal pumping rates in treated worms. Overall, CSS supplementation under glucose stress conditions modulated the expression of these key genes, suggesting that CSS influences lipid metabolic pathways in C. elegans, potentially improving lipid metabolism and reducing the negative effects of glucose-induced stress.

Figure 9. Expression of (a) sams-1, fat-7, fasn-1 sbp-1 and pod-2 genes involved in fat synthesis and (b) cpt-1, hacd, acs-2, tph-1, nhr-49, atgl-1, ser-6 involved in lipid breakdown, and ser-1 involving in pumping rate, in 1-day-old adults treated or not with 250 µg/mL or 25 µg/mL extracts and grown on HGD or under standard conditions (control). Statistical analysis was performed using two-way ANOVA followed by the Bonferroni post-test. Experiments were performed in triplicate (200 worms for each condition). Data are presented as mean ± SD (** p < 0.01, *** p < 0.001, ns: not significant).

2.9. CSS Activates DAF-16 Translocation and Reduces Lipid Accumulation in Mutant Worms

It has been reported that glucose is a potent lifespan-shortening agent in C. elegans, down-regulating the activities of the lifespan-extending proteins DAF-16/FOXO [44]. To assess whether CSS modulates the DAF-16/FOXO pathway, we examined nuclear localization of daf-16::GFP. Under basal conditions, CSS supplementation induced moderate DAF-16 nuclear translocation compared to untreated controls, indicating a mild activation of this stress-responsive transcription factor (Figure 10a). Upon exposure to heat stress (1 h at 37 °C), worms treated with CSS displayed significantly enhanced DAF-16 nuclear localization relative to stressed controls, demonstrating that CSS amplifies DAF-16–mediated stress response. Specifically, 250 µg/mL CSS treatment led to a 15% increase in the percentage of DAF-16 nuclear translocation, observed under both non-stressed and heat-stressed conditions (Figure 10b). Next, we investigated the role of DAF-16 in lipid metabolism by analyzing lipid accumulation, via RT-qPCR and BODIPY staining, in daf16(mu86) loss-of-function mutants. Similarly in the wild-type counterpart, 250 µg/mL CSS supplementation in HGD condition significantly upregulated the expression of ser-4 and ser-6 genes compared to the standard diet control, indicating an improved lipid breakdown capacity (Figure 11a). Intriguingly, CSS treatment significantly reduced lipid accumulation in these mutants, to an extent comparable to that observed in wild-type N2 worms (Figure 11b).

Figure 10. Fluorescence analysis of the daf-16::GFP transgenic strain at the 1-day-old adult stage following supplementation with 250 µg/mL CSS. (a) Representative fluorescence images showing DAF-16::GFP localization. Scale bar = 100 µm. (b) Percentage of worms displaying GFP-positive nuclei. Statistical analysis was performed using one-way ANOVA followed by the Bonferroni post-test. Data were obtained from three independent experiments (20 worms per condition) and are expressed as mean ± SD (* p < 0.05). The red asterisk indicates statistical significance compared to the HGD group without stress, whereas the green asterisk indicates statistical significance compared to the HGD group subjected to thermal stress (37 °C for 1 h).

Figure 11. (a) Expression of tph-1, ser-4 and ser-6 in daf-16(mu86) mutant worms at the stage of 1-day of adults. Statistical analysis was performed using two-way ANOVA followed by the Bonferroni post-test. Experiments were performed in triplicate (200 worms for each condition). Data are presented as mean ± SD (** p < 0.01; ns: not significant). (b) BODIPY™ 493/503 staining of lipid droplets in 1-day-old mutant nematodes fed OP50 and supplemented with different concentrations of CSS. Scale bar: 50 μm. Experiments were performed in triplicate (10 worms for each condition).

2.10. CSS Restores Lifespan in nhr-49 Mutant Worms

Given the role of NHR-49 in mediating metabolic balance, as observed in previous results, we investigated the effects of CSS supplementation in nhr-49 mutant worms, which exhibit an obese phenotype due to impaired lipid metabolism. Unlike wild-type worms, where CSS had no significant impact on longevity, its effects in nhr-49 mutants suggest a compensatory mechanism in response to metabolic dysfunction (Figure 12). Lifespan analysis was conducted under standard dietary conditions, as nhr-49 mutants inherently accumulate excessive fat. As shown in Figure 12a and Table S2, untreated nhr-49 mutants exhibited a marked reduction in lifespan, with 50% survival occurring by day 5. However, supplementation with 25 µg/mL CSS extended 50% survival to day 7, while 250 µg/mL CSS further extended it to day 8, indicating a dose-dependent protective effect. The restoration of lifespan highlights CSS’s role as a metabolic modulator, improving lipid utilization and counteracting obesity-associated stress. Lipid accumulation was further evaluated in nhr-49 mutant worms by BODIPY™ 493/503 staining (Figure 12b). Untreated mutants exhibited intense fluorescence, consistent with their known obese phenotype. CSS supplementation markedly reduced fluorescence intensity, indicating a decrease in lipid droplet content and suggesting that CSS can alleviate fat accumulation even in the absence of functional NHR-49.

Figure 12. (a) Kaplan–Meier survival plot of nhr-49 mutants fed with heat-killed OP50 bacteria and supplemented with CSS at concentrations of 250 µg/mL or 25 µg/mL, under standard condition. For each data point, n = 80 per experiment (*** p < 0.001). The lifespan assay was performed in triplicate, using worms fed with OP50 as a reference (untreated). (b) BODIPY™ 493/503 staining of lipid droplets in nhr-49 mutant worms supplemented with 250 µg/mL CSS, as compared with untreated (control). Scale bar: 50 µm. n = 20 per each condition. Data are representative of three independent experiments.

3. Discussion

The findings of this study suggest that dietary supplementation with CSS can attenuate obesity-related conditions, such as lipid droplet accumulation and the dysregulation between lipogenesis and β-oxidation. These protective effects may be attributed to the modulatory role of CSS on serotonin metabolism, thereby supporting the proposed hypothesis (Figure 13). CSS, a by-product of coffee roasting, is rich in bioactive compounds, particularly antioxidants such as chlorogenic acid and caffeine, as well as dietary fibers. The chemical composition of the CSS extract used in this study has been previously characterized in our laboratory and reported in detail by Nolasco et al. [4], making it a valuable resource for industries such as food, cosmetics, and pharmaceuticals [45,46].

Figure 13. CSS promotes healthspan in C. elegans by enhancing antioxidant defenses via the DAF-2/DAF-16 pathway and modulating lipid metabolism. CSS supplementation restores lipid homeostasis under high-glucose conditions through nhr-49 activation and supports serotonin-mediated fat regulation.

This study explored the effects of various concentrations of CSS extracts on biological parameters in the Caenorhabditis elegans model. While no significant impact on the lifespan of nematodes was observed, CSS demonstrated the ability to modulate healthspan, particularly by influencing larval development and oxidative stress responses. Specifically, CSS at 25 μg/mL significantly increased nematode body length at both 4 and 7 days post-hatching, indicating enhanced larval development. This index serves as an indicator of improved overall health, as larger body size is often correlated with better physiological conditions [47]. Additionally, while CSS initially elevated reactive oxygen species (ROS) levels in young nematodes, a significant reduction in ROS was observed in older worms. The early increase in ROS in young CSS-treated worms, which was not associated with toxicity, may reflect an adaptive response to stress. Transient ROS elevations can function as signaling cues that activate endogenous defense mechanisms, potentially contributing to the reduced oxidative stress observed at later stages [48,49].

These antioxidant effects could be attributed to CSS’s chemical composition, which includes bioactive compounds known for their protective properties against oxidative damage. The IR spectrum of coffee silverskin (CSS powder) reveals the typical absorption bands of lignocellulosic materials. This observation is consistent with previous reports, which describe its composition as being mainly composed of carbohydrates, principally cellulose and hemicellulose, and proteins, together with lignin, fatty acids, polyphenols, minerals, and other minor organic and inorganic constituents [15,16,50,51]. The comparison between the infrared spectra of CSS powder and its hydroalcoholic extract at the concentration of 250 µg/mL (CSS extract) highlights the transition from a lignocellulosic material to a phenolic-rich extract. The disappearance of polysaccharide-related bands and the relative increase in intensity of carbonyl and aromatic-associated peaks in the spectrum of CSS extract indicates that the hydroalcoholic extraction selectively enriched polyphenolic constituents from the CSS, in agreement with previous reports [52,53]. Subsequent characterization of hydroalcoholic extracts through ESI-MS/MS confirmed this observation, revealing the presence of xanthines (i.e., caffein), and phenolic markers, including hydroxycinnamic acids derivatives (chlorogenic acid and di-caffeoyl quinic acid, among others), in line with previous findings [54].

The presence of such phenolic compounds supported our hypothesis of an antioxidant effect of CSS, as further corroborated by real-time qPCR analysis that revealed an upregulation of detoxifying genes, including gst-4 and sod-3. Two key signaling pathways, the insulin/IGF-1 signaling (IIS) and mitogen-activated protein kinase (MAPK) pathways, play primary roles in regulating the activity of antioxidant enzymes, the main force of the antioxidant defense system, in response to oxidative stress in C. elegans [55]. Nuclear localization of the core transcription factors DAF-16 and SKN-1 (Skinhead-1) within the IIS and MAPK signaling pathways increases in response to elevated ROS levels and oxidative stress in worms [56]. These factors bind to downstream antioxidant response elements, which function as crucial signals leading to the production of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), to help detoxifying and protecting against oxidative damage [57].

Summarizing, the early, non-toxic rise in ROS likely triggers a systemic adaptive response, in which the DAF-2/DAF-16 pathway acts as an antioxidant defence, that will help the nematode maintain metabolic stability during prolonged glucose stress. These findings are consistent with previous studies showing that chlorogenic acid can enhance antioxidant defenses in C. elegans, promoting stress resistance and longevity [58]. Moreover, CSS did not significantly alter nematode locomotion, as measured by body bending, but it enhanced the pumping rate, an indicator of food intake. This suggests a beneficial effect on muscular function and vitality in aged nematodes, indicating that CSS may help maintain muscle integrity, particularly in older individuals, thus contributing to an extended healthspan.

Given the highly conserved pathways of energy homeostasis between mammals and C. elegans, this nematode represents a robust model for investigating how functional ingredients modulate fat storage, typically assessed through the quantification of triglyceride accumulation [59]. In our study, CSS treatment effectively counteracted HGD-induced lipid imbalance in a dose-dependent manner. This anti-obesity effect was phenotypically confirmed by both BODIPY 493/503 and Oil Red O (ORO) staining: while BODIPY highlighted a reduction in the number and size of lipid droplets, ORO analysis, specifically targeting neutral lipids and triglycerides, showed a parallel decline in total lipid stores. These observations align closely with our molecular findings, where the significant downregulation of fasn-1 (fatty acid synthase) and fat-7 (Δ9-desaturase) provides a mechanistic explanation for the leaner phenotype. Indeed, these enzymes are pivotal for de novo lipogenesis and fatty acid desaturation; in particular, the inhibition of fat-7 limits the pool of monounsaturated fatty acids (MUFAs) available for triacylglycerol assembly [60,61].

Interestingly, the biological response to CSS followed a non-linear pattern. While intermediate and high doses (25 and 250 µg/mL) successfully mitigated glucose toxicity, the lowest concentration (2.5 µg/mL) slightly reduced lifespan compared to the HGD control. This could suggest an hormetic effect, where only specific concentrations trigger the adaptive stress-response pathways (such as DAF-16 or SKN-1) necessary to enhance fitness [62]. At 2.5 µg/mL, CSS likely acts as a sub-threshold stimulus, failing to activate these protective mechanisms and leaving the organism vulnerable to the pro-oxidant effects of a high-glucose diet [63]. The convergence of histochemical, fluorescence-based, and transcriptional data provides a robust evidence base for the role of CSS in suppressing lipid storage and restoring metabolic balance [64,65].

Moreover, it has been observed that the metabolic effects exerted by CSS in C. elegans are mediated by NHR-49 and SER-6. NHR-49, the worm homolog of mammalian PPARα, is a central regulator of fatty acid β-oxidation, lipid homeostasis, and energy balance [66]. Its upregulation following CSS supplementation, particularly in HGD conditions, suggests that CSS enhances lipid catabolism, promoting a metabolic shift toward increased energy utilization. Notably, in nhr-49 mutant worms, which display an obese phenotype due to defective lipid oxidation, CSS restored lifespan in a dose-dependent manner. This context-dependent response may explain why CSS rescues lifespan in metabolically compromised nhr-49 mutants but does not extend lifespan in wild-type worms, where lipid homeostasis is already efficiently regulated. This effect can be likely driven by enhanced β-oxidation, as observed in HGD-fed animals in which the upregulation of acs-2 and hacd compensate for the metabolic imbalance that is typically observed in these mutants.

Additionally, our findings highlighted a potential link between serotonin signaling and lipid metabolism, with SER-6 emerging as a key downstream effector of CSS-induced metabolic adaptations. SER-6, an octopamine receptor involved in the positive regulation of β-oxidation [41], was significantly upregulated in worms treated with 250 µg/mL CSS. Since SER-6 is regulated by TPH-1, the rate-limiting enzyme in serotonin biosynthesis, and TPH-1 itself showed a partial recovery following CSS treatment, we hypothesize that CSS may influence lipid metabolism via serotoninergic modulation. A similar upregulation was observed in daf-16(mu86) mutants, indicating that lipid-lowering effect is independent of DAF-16 function. CSS activates DAF-16 translocation under both basal and stress conditions, but it was not involved in lipid breakdown, suggesting that the extract could exert beneficial metabolic effects via additional pathways.

Taken together, these results indicate that CSS exerts its metabolic effects through a dual mechanism: the direct activation of lipid oxidation pathways via NHR-49, and serotonin-mediated regulation of β-oxidation via the TPH-1/SER-6 axis. Serotonin plays a crucial role in regulating feeding behavior and metabolic homeostasis in C. elegans [43]. The increased expression of ser-4 after CSS treatment suggests an enhancement of serotonin-mediated signaling, which could explain the observed rise in pharyngeal pumping rates. These findings provide further evidence that CSS acts as a metabolic modulator, improving lipid utilization and restoring energy balance in conditions of metabolic stress (Figure 12). The coordinated response involving both NHR-49 and serotonin signaling aligns with the principle of hormesis [67,68], suggesting that CSS induces a mild physiological stimulation that effectively ‘primes’ the organism to maintain homeostasis under metabolic pressure.

The beneficial properties of foods on health also include the possible prebiotic effect on the intestinal microbiome. A prebiotic is defined as a substrate that is selectively used by host microorganisms to confer a health benefit [69]. Dietary fibers and other non-digestible carbohydrates that reach the intestine serve as substrates for bacterial fermentation and therefore can influence the composition and metabolic activities of the intestinal microbiota [70]. The chemical composition of CSS, depending on the geographical origin and the method of processing and roasting, indicates a fiber content of 34–68%, of which 11% represents soluble fiber and 46–56% insoluble fiber [4,32,71]. The results of our analysis revealed the presence of several polysaccharide compounds in CSS powder, supporting CSS as a valuable source of prebiotic compounds [72]. Several studies indeed indicated that CSS may promote the growth of bifidobacteria while inhibiting the growth of Bacteroides and clostridia, which further supports its potential as a prebiotic source in food formulations [72,73,74]. Notably, CSS has already shown the ability to induce adaptations in the gut microbiota in other simplified animal models (Hermetia illucens larvae), where the microbiota composition significantly changed upon rearing on CSS. This adaptability implies that CSS may offer substrates that selectively sustain microbial populations [75]. In the present work, we evaluated the ability of CSS to support the growth of four commercial probiotic strains, by comparing their growth in the presence of this matrix as the only carbon source with the growth in the presence of glucose. Overall, the results suggest the possible prebiotic potential of CSS; however, this needs to be confirmed through further analyses in more complex experimental models.

A distinctive strength of our study lies in demonstrating that the beneficial effects of CSS in C. elegans extend considerably beyond its well-established antioxidant capacity. In contrast to previous research, which primarily attributed the bioactivity of CSS to direct free radical scavenging, our data reveal a novel and active role in reprogramming host lipid metabolism. Crucially, we identified that this regulation is not passive but rather mediated by specific, conserved genetic pathways involving NHR-49 and serotonin, redefining CSS not merely as a redox-active agent, but as a versatile metabolic modulator capable of influencing host health through synergistic interactions among nuclear receptors and neuroendocrine signaling.

4. Materials and Methods

4.1. ATR-FTIR Spectroscopy

The chemical nature of CSS powder and its hydroalcoholic extract at the concentration of 250 µg/mL (CSS extract) were analyzed by Attenuated Total Reflectance–Fourier-Transform Infrared Spectroscopy (ATR-FTIR). Spectra were acquired on dry CSS powder and CSS extract using a Vertex 70 spectrometer (Bruker Optics, Gmbh, Ettlingen, Germany) equipped with a single reflection Diamond ATR cell, a standard MIR source (HeNe) and a room temperature DTGS detector. The spectra were recorded with 64 scans in the mid-infrared range (400–4000 cm⁻¹) at a resolution of 4 cm⁻¹.

4.2. Mass Spectrometry Analysis

Silverskin extract was analysed using an API 3200 QTRAP mass spectrometer (Applied Biosystems, Foster City, CA, USA) equipped with an electrospray ionization (ESI) source. Extract was directly infused into the ESI source at a flow rate of 10 µL/min using a syringe pump. Spectra were acquired in both positive and negative ion modes. The ion spray voltage was set at −4500 V in negative mode and +4500 V in positive mode. The source temperature was maintained at 300 °C. Nitrogen was used as curtain gas (20 arbitrary units), ion source gas 1 (GS1, nebulizer gas) and ion source gas 2 (GS2, auxiliary gas) at 20 and 25 arbitrary units, respectively. Full-scan mass spectra were acquired in the m/z 100–800 range. The most abundant ions detected in the ESI-MS profiles were selected as precursor ions and subjected to MS/MS experiments (a representative portion of the full-scan and the MS/MS spectra of selected ions are reported in Figure S2). Product ion spectra were obtained by collision-induced dissociation (CID) using nitrogen as collision gas, with collision energies ramped from 5 to 50 eV under both positive and negative ESI conditions. The resulting MS/MS spectra were compared with those of authentic standards, when available, or with fragmentation patterns reported in the literature. Data acquisition was performed using Analyst software (version 1.5.1, Applied Biosystems).

4.3. Assessment of Prebiotic Activity

The potential prebiotic effect of the sterilized coffee silverskin (CSS) powder was determined by screening its impact upon the growth of four commercial probiotic strains: Lacticaseibacillus rhamnosus GG^® (AG Pharma, Rome, Italy); Limosilactobacillus reuteri DSM17938^® (Noos, Rome, Italy), Lacticaseibacillus paracasei DG11572^® (Alfasigma, Bologna, Italy), Bifidobacterium longum W11^® (PharmExtracta, Piacenza, Italy). All the strains were used as monoculture and, before the assays, were grown in MRS broth (Oxoid) at 37 °C for 24 h under anaerobic conditions. For the fermentation assays, each probiotic strain was inoculated in MRS medium without glucose (MRSnoG, Liofilchem, Italy) alone, or with the addition of either glucose, inulin (used as a positive controls), or sterilized CSS powder, each at a concentration of 2% (w/v), in separate tubes. The MRSnoG medium served as the negative control.

To determine the viable bacterial counts, 1 mL aliquots of bacterial cultures were collected before and after fermentation (0 h and 24 h), serially diluted in 0.9% NaCl, plated on MRS agar and incubated at 37 °C for 24 h under anaerobic conditions. Before fermentation assay, CSS powder was also examined for the presence of bacteria by an estimation of total viable counts (TVCs) on plate count agar (PCA; Oxoid) after 24 h of incubation at 30 and 37 °C under aerobic condition, and for Enterobacteriaceae on violet red bile glucose agar (VRBGA, Oxoid) after 24 h of incubation at 37 °C.

4.4. Assessment of Antimicrobial Activity

The potential antimicrobial activity was evaluated by the spot-on agar test, in which 5 uL of sterilized CSS powder at a concentration of 2% (w/v) was spotted onto Tryptone Soy Agar (TSA, Oxoid) plates previously inoculated with 1 × 10⁶ CFU/mL indicator strains in the exponential growth phase. The pathogens used in the test were Salmonella Thiphymurium LT2; Salmonella Give, Salmonella Derby, Salmonella Enteritidis, Salmonella New Port (provided by the Istituto Zooprofilattico Sperimentale del Mezzogiorno (Portici, Naples, Italy); Listeria monocytogenes OH; Listeria monocytogenes CAL; Listeria monocytogenes SA; Listeria innocua 1770 (provided by the CREA Research Centre for Animal Production and Aquaculture, Lodi, Italy); and ETEC K88 (provided by the Lombardia and Emilia Romagna Experimental Zootechnic Institute, Reggio Emilia, Italy). The spotted plates were then incubated for 18 h under optimal growth conditions and the inhibition zones (radii of microbial growth inhibition, halos) around the wells were measured in millimetres (mm) according to Balouiri et al. 2016 [76]. In the same plates, the test efficacy was confirmed by adding 2 µL of 32 g/mL chloramphenicol (Sigma-Aldrich, St. Louis, MO, USA). The antimicrobial capacity was expressed as the radius of the inhibition halo (mm).

4.5. C. elegans Strain and Lifespan Assay

The wild-type C. elegans strain, Bristol N2, CF1038 daf-16(mu86), zIs356 [daf-16p::daf-16a/b::GFP + rol-6 (su1006) and STE68 [nhr-49(nr2041)/I] were grown at 16 °C on Nematode Growth Medium (NGM) plates seeded with E. coli OP50, which was killed at 65 °C for 90 min as described in [33]. Fertile adults were placed to lay embryos for 8 h on NGM plates seeded with 30 µL of heat-killed E. coli OP50 and three different concentrations of hydro-alcoholic extracts: 2.5 µg/mL, 25 µg/mL, and 250 µg/mL. The hydroalcoholic extracts were prepared suspending 10 mg of CSS in 40 mL of EtOH:H₂O 80:20. When the progeny reached fertility (t0), 80 worms per condition were transferred to new plates with the same conditions and monitored daily. A worm was considered dead when it did not respond to touch. Adults were transferred daily to avoid progeny contamination. To mimic a high-glucose diet (HGD), 2% glucose was added to the agar and salts mix of the NGM, and experiments were performed at 20 °C. All lifespan assays were performed in triplicate.

4.6. Brood Size and Body Length

Age-synchronized N2 worms were obtained by allowing fertile adults to lay eggs on NGM plates seeded with bacteria at 16 °C. For fertility assessment, ten individual worms per condition were transferred daily onto fresh plates throughout the reproductive period, and the total number of progeny was quantified using a stereomicroscope, following established procedures [55]. Each experiment was conducted in triplicate.

Body length measurements were performed on worms from day 1 to day 5 after egg hatching. Animals were imaged using a Zeiss Axiovert 25 inverted microscope equipped with an Axiocam 208 camera (Carl Zeiss AG, Oberkochen, Germany). Worm length was quantified using ZEN 11 software and expressed relative to untreated control worms. For each experimental condition, at least 30 animals were analyzed, and measurements were obtained from a minimum of three independent experiments.

4.7. Aging Markers’ Analysis

To assess the pharyngeal pumping rate, the number of grinder contractions was counted under a stereomicroscope in 10-day-old adult worms that had been fed the three different concentrations of CSS extracts since embryo hatching, in standard or in HGD condition. Ten worms were observed for each treatment over a 30 s period. The experiments were repeated in triplicate. The locomotion ability of the nematodes was evaluated by counting body bends over 30 s. Specifically, as described in [77], 10 worms per treatment were washed in M9 buffer to remove bacteria and then placed in 40 μL of M9 buffer to facilitate the measurement of locomotion. The experiments were performed in triplicate.

4.8. Measurement of Intracellular ROS

Intracellular ROS levels were measured using the fluorescent probe 2′,7′-dichlorofluorescein diacetate (H₂DCFDA), following a previously described protocol [78] with minor modifications. Age-synchronized N2 worms were exposed to 25 μg/mL CSS starting from embryo hatching. At the stage of 1-day-old adulthood, worms were washed three times with M9 buffer to remove residual bacteria. For each condition, 50 worms were transferred to a 96-well microtiter plate, and H₂DCFDA was added to a final concentration of 50 μM. Fluorescence was measured using a multiplate reader (GloMax multidetection system, Promega, Madison, WI, USA) at excitation/emission wavelengths of 485/520 nm. Each experiment was performed in triplicate, and fluorescence values were normalized to the corresponding untreated control.

4.9. Real-Time qPCR

Real-time quantitative PCR analysis was performed on age-synchronized 1-day-old adult worms. For each experimental condition, approximately 200 animals were collected and lysed for total RNA extraction, following a previously established protocol [38]. Transcript levels of genes involved in the oxidative stress response (daf-2, daf-16, sod-3, and gst-4) were analyzed in worms maintained under standard dietary conditions and supplemented with CSS at 25 µg/mL. The same procedure was applied to assess the expression of genes related to lipid metabolism in nematodes grown under high-glucose diet (HGD) conditions and supplemented with CSS at 25 µg/mL and 250 µg/mL. The lowest concentration tested in other assays (2.5 µg/mL) was not included in RT-qPCR analyses, as it did not elicit measurable physiological effects. Untreated worms grown under standard dietary conditions were used as controls. Relative gene expression was calculated by normalizing the mean Ct value of each target gene to that of the housekeeping gene act-1. The primers sequences used for Real-Time qPCR are listed in Table S1.

All experiments were performed in triplicate.

4.10. Lipid Droplet Visualization and Triglycerides Quantification

Lipid droplet accumulation was evaluated in age-synchronized 1-day-old adult C. elegans fed heat-killed E. coli OP50 and exposed to the indicated CSS concentrations (2.5, 25, or 250 µg/mL) under HGD conditions. Untreated worms maintained under standard dietary conditions were used as controls. Following treatment, animals were washed three times with M9 buffer to remove residual bacteria. Neutral lipid droplets were visualized using BODIPY™ 493/503 (Life Technologies, Carlsbad, CA, USA), following a previously described protocol [33]. Stained worms (n = 30 for each condition) were mounted on 3% agarose pads containing 20 mM sodium azide and imaged by fluorescence microscopy using a Zeiss Axiovert 25 microscope at 32× magnification.

In parallel, neutral lipid and triglyceride accumulation was assessed using Oil Red O staining (Sigma-Aldrich), according to [79]. After staining, worms were mounted on agarose pads. Oil Red O–stained worms were imaged under bright-field microscopy using the same microscope and magnification settings.

For both staining methods, lipid content was quantified by ImageJ (version 1.54K)-based analysis of staining intensity, calculated as the ratio of stained pixels to total worm area. For each experimental condition, ten worms were analyzed, and mean values were reported. Each experiment was performed in triplicate.

4.11. Fluorescence Analysis in the Daf-16::GFP Transgenic Strains

At the 1-day adult stage, synchronized daf-16::GFP transgenic worms fed heat-killed OP50 and 250 µg/mL CSS from embryo hatching were anesthetized with sodium azide (20 mmol L⁻¹) (Sigma-Aldrich, St. Louis, MO, USA) and observed using a Zeiss Axiovert 25 microscope. Experiments were performed in triplicate, with 20 worms per condition in each replicate. Images were acquired with an exposure time of 0.2 s, and fluorescence was analyzed using ImageJ software. Scale bars were added using Zeiss ZEN Microscopy Software 2011.

4.12. Statistical Analyses

All experiments were performed in at least three independent replicates, with the number of individuals and experimental conditions specified in the figure legends or tables. Data are presented as mean ± SD. Statistical significance was determined using Student’s t-test, one-way ANOVA or two-way ANOVA followed by either Bonferroni or Tukey’s honestly significant difference (HSD) post hoc test, as indicated (GraphPad Prism 9.0, GraphPad Software Inc., La Jolla, CA, USA). Longevity analyses were conducted using the Kaplan–Meier method, and differences in survival curves were assessed with the log-rank (Mantel–Cox) test. Differences with p-values < 0.05 were considered significant and indicated as follows: * p < 0.05, ** p < 0.01, and *** p < 0.001.

5. Conclusions

Taken together, the results reported in the present work highlighted that CSS improves C. elegans metabolism through two main mechanisms: direct activation of lipid oxidation via NHR-49 and serotonin-mediated regulation of β-oxidation via the TPH-1/SER-6 axis. The increase in ser-4 expression suggests enhanced serotonin signaling, which likely drives the observed rise in pharyngeal pumping, consistent with the know role of serotonin in feeding and metabolic homeostasis. In this framework, CSS could act as a hormetic modulator, leveraging endogenous stress-response pathways to optimize energy utilization and improve overall metabolic fitness. Moreover, CSS promoted the growth of probiotic strains, suggesting potential prebiotic properties. Overall, these findings therefore identify CSS as a metabolic modulator capable of alleviating oxidative and metabolic stress, supporting its sustainable application in the development of functional foods and nutraceuticals.