A Whole Tomato Food Supplement Modulating Lipidomic and Proteomic Profiles in HepG2 Cells as a Dietary Tool for the Management of Non-Alcoholic Fatty Liver Disease

1. Introduction

Epidemiological and experimental evidence increasingly supports the protective role of dietary antioxidants on fat liver accumulation, generating oxidative stress [1,2] lipid peroxidation, mitochondrial dysfunction, and inflammation [3,4,5,6].

Standing the dietary origins of non-alcoholic fatty liver disease (NAFLD) [7] and suggested by the results of in vitro/vivo models, functional nutrients and foods [8] capable of modulating fat metabolism and associated inflammation, are currently undergoing scrutiny since they may represent variably efficient, reversible inhibitors, with low toxicity, contained costs, thus available to large populations fractions. In this regard, increasing attention has been focused on the polyene lycopene, potent antioxidant of the carotenoid family, which through the induction of other endogenous antioxidants [9], possesses anti-inflammatory [10] and lipid-lowering properties [11], which are shared by its metabolites [12]. In this context, it should be underlined that the lycopene hypolipidemic activity relies on the dual mechanism of HMG-CoA reductase inhibition and downregulation of PCSK-9 mRNA synthesis [13].

Because of its hepatic accumulation [14], where active metabolites, i.e. apo-lycopenals and apo-lycopenones are generated [15], the mitigating effects of lycopene and lycopene-containing foods are attractive candidates to exploit dietary tools of wide acceptance to control the development and progression of NAFLD, a disease of increasing incidence and prevalence [16,17].

To acquire the broad spectrum of its healthy biological properties, the trans isomeric form of the naturally occurring lycopene needs to be modified, by the digestive processing [18], in the cis configuration the only biologically active isomer [19] and by the concomitant uptake of other tomato micronutrients, such other carotenoid [20] and nutrients generated by the cooking of the berry [21]. Indeed, the consumption of whole fruits has been shown to result in healthier effects than single lycopene supplementation, as demonstrated in laboratory [16] and clinical studies [22,23]. Therefore, the unique combination of antioxidant and anti-inflammatory nutrients [24] with converging biological activities of the berry [25] advocates the choice of whole tomato dietary consumption as a functional food for equitable and sustainable healthy diets [26]. Along this line of investigation, a novel whole tomato (98%) food supplement (WTFS) [27], enriched with olive waste water (2%) food in the form of an additives/ excipients-free powder [28], has been recently described which is characterized by a multinutrient composition [29], capable of interfering with metabolic pathways sustaining both inflammation and neoplastic transformation [30].

This improved functional food may be a candidate for further testing to mitigate the incidence of lipid accumulation-related liver diseases. To test this possibility, we have performed a lipidomic and proteomic analysis on HepG2 human liver cells exposed to the WTFS.

2. Materials and Methods

2.1. Cell Cultures

The HepG2 human hepatoblastoma cells, a reference target to study liver function and hepatotoxicity [31], obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), were plated at 1×10⁶ in T-75 cell culture flasks in minimum Essential Media supplemented with 10% heat-inactivated fetal calf serum containing 2 mmol/L L-glutamine, 100 IU/mL penicillin, 100 mg/mL streptomycin, 2.2 mg/L sodium bicarbonate, and 1 mmol/L sodium pyruvate, under a humidified atmosphere of 95% air/5% CO2 at 37° C, as previously descrided [32]. WTSF preparation in dimethyl sulfoxide (DMSO) and cells treatment followed the conditions described by Rubini et al. [33], and HepG2 cells cultured with only DMSO were used as control (CTRL). Cells were collected as a dry pellet following washing with phosphate-buffered saline and subsequent counting. A precipitation solvent composed of water-saturated butanol and 20 mM ammonium acetate in methanol (in a 1:1 volume ratio) was added to achieve a concentration of 3,500 cells/µL. The solution was then sonicated and centrifuged for 5 minutes at 4,500 rpm at room temperature. The supernatant was then frozen at -80° C for later lipidomic analysis.

2.2. Lipidomic Analysis

Lipidomic analysis was conducted using a 5500 QTRAP LC-MS/MS system (AB Sciex, Framingham, MA, USA) equipped with an electrospray ionization source and coupled with an ExionLC HPLC system (AB Sciex). Samples were injected onto an Xbridge BEH C18 precolumn (3.5 µm, Waters Corporation, Milford, MA, USA) and subsequently separated using an analytical column, Xbridge C18 (3.5 µm, 2.1 x 100 mm, Waters Corporation). The injection volume was 1 µL for positive ion mode and 5 µL for negative ion mode. The column temperature was set at 50°C, and elution was conducted at a flow rate of 0.400 mL/min by incrementally increasing the concentration of organic solvent B from 0% to 97% over 50 minutes. Solvent A was composed of 10 mM ammonium formate in a mixture of water, acetonitrile, and 2-propanol (50:30:20 v/v/v), while solvent B contained 10 mM ammonium formate in a mixture of water, acetonitrile, and 2-propanol (1:9:90 v/v/v). All samples were analyzed in triplicate in both positive and negative modes.

The multiple reaction monitoring analysis involved the detection of 333 transitions in positive ion mode, encompassing various lipid classes, including carnitines, cholesterol esters (CE), ceramides (Cer-d), cholesterol, diacylglycerols (DG), glucosylceramides (GCer), lactosylceramides (LacCer), lysophosphatidylserines (LPS), lysophosphatidylcholine (LysoPC), lysophosphatidylethanolamines (LysoPE), phosphatidylcholines (PC), phosphatidylethanolamines (PE), phosphatidylserines (PS), sphingomyelins (SM), and triacylglycerols (TG). In negative ion mode, 93 transitions were monitored, which included bile acids (BA), fatty acids, cardiolipins (CL), lysophosphatidic acids (LPA), lysophosphatidylinositol acids (LPI), phosphatidic acids (PA), phosphatidylinositol acids (PI), phosphatidylglycerol acids (PG), and sulfatides (Sul-d).

Data processing was conducted using MultiQuant software version 3.0.2 (AB Sciex), and statistical analysis was performed utilizing the R software package (version 4.3.2).

2.3. Olink Analysis

The analysis was conducted by using the Proximity Extension Assay Olink Target 48 Cytokine panel (Olink Proteomics, Uppsala, Sweden), providing absolute (pg/mL) measurements for the selected cytokines. A comprehensive list of the 45 analysed proteins, along with their respective acronyms and UniProt codes, is provided in Supplemental Table S1. This approach uses specific antibody probes marked with dual oligonucleotides that bind to target proteins. Quantitative DNA detection follows, where the oligonucleotide sequence is amplified via microfluidic real-time PCR. Quality control procedures and normalization were performed on cycle threshold data from both internal and external controls.

2.4. Gene Ontology Analysis

Protein-protein interaction analysis and functional enrichment for Gene Ontology (GO) categories were performed using the STRING database v12.0 (https://string-db.org/). A set of input proteins was analyzed with the interaction confidence score set to default (medium confidence ≥0.4), and the maximum number of interactors was limited to no more than 5 to ensure a functionally relevant and interpretable network. Functional enrichment was evaluated under the GO Molecular Function category. The significance of enrichment was assessed using STRING’s built-in statistical framework, based on a modified Fisher’s exact test corrected for multiple testing (false discovery rate).

3. Results

Targeted lipidomic analysis in positive ion mode was performed to assess the impact of WTFS treatment on the lipid composition of HepG2 cells. Figure 1 displays a bubble plot summarizing fold changes in lipid species abundance. Each bubble represents an individual lipid species, colored by lipid class. The x-axis denotes the fold change, while bubble size is proportional to statistical significance, represented as 1 minus the p-value. This visualization allows simultaneous assessment of both the magnitude and significance of lipid alterations across different classes.

As illustrated in Figure 1, WTFS exposure resulted in a significant reduction in the relative abundance of multiple lipid classes compared to DMSO-treated cells (CTRL). Specifically, the levels of CE, DG, LysoPC, LysoPE, PE, and TG were decreased. Among these, TG and CE exhibited the most pronounced reduction, with an average fold change of 0.71 and 0.56, respectively. Conversely, a significant increase was observed in the levels of GCer, which displayed a mean fold change of 1.40. No appreciable alterations were detected in the abundance of carnitine, PC, or SM, indicating a selective remodeling of the lipid profile upon WTFS treatment.

In negative ion mode, treated cells with WTFS showed a decrease in the levels of fatty acids and LPI compared to CTRL (Figure 2). Conversely, only CL and PG exhibited slight increases in the WTFS-treated cells. There were no measurable changes in the relative abundances of BA, PA, LPA, PI, and Sul-d.

Since cytokines and growth factors, known mediators of liver function, can contribute to the onset and progression of various liver diseases [34], we investigated the cytokines’ modulatory activity on HepG2 cells treated with WTFS.

According to the proteomic analysis (Figure 3), three molecules out of the 45 proteins analyzed (see Table S1) exhibited significant downregulation by the WTFS: a) TGFA, the epidermal growth factor (EGF) family member known as transforming growth factor-α (TGF-α), which plays a crucial role in modulating cell growth, differentiation, migration, and survival [35]; b) TNFSF12, also referred to as TNF-related weak inducer of apoptosis (TWEAK), a multifunctional cytokine with a diverse array of biological activities [36], which also serves as a ligand for the fibroblast growth factor-inducible 14 (Fn14) receptor; c) FLT3LG, or Fms-related tyrosine kinase 3 ligand, that by binding the Flt3/CD135 receptor, induces dimerization and autophosphorylation of the receptor, and activates multiple downstream signaling pathways, including PI3K/Akt/mTOR, JAK/STAT, and RAS/RAF/Erk. These pathways are involved in the survival and proliferation of various cell lineages, including hepatocytes [37].

The STRING network analysis, which identified functionally enriched molecular functions among the input proteins (max 5 interactors) showed two enriched GO Molecular Function terms: “cytokine activity” (GO:0005125) indicating significant involvement of several nodes in cytokine-mediated signaling and “receptor ligand activity “(GO:0048018), representing proteins with potential to act as ligands for receptor-mediated processes (Figure 4).

4. Discussion

NAFLD [17] is becoming globally a highly prevalent disease in the general population [38], especially in those patients affected by type 2 diabetes mellitus and obesity [39]. Of relevance, NAFLD is also the most frequent pediatric liver disease [40,41]. Although no specific drug is currently available for NAFLD, this disease may be amenable to preventive, therapeutic interventions through the adherence to international guidelines recommending a lifestyle-based approach, relying on healthy diets [42]. This can modulate hepatic steatosis and counteract liver damage, preventing and delaying the evolution of NAFLD to cirrhosis and cancer [43]. In this context, phytochemical and natural compounds are undergoing an in-depth investigation [44]. Carotenoid are of major interests [45] and multidisciplinary evidences strongly indicate that lycopene [10], a potent and largely available dietary antioxidant/anti-inflammatory nutrient, may be a potential candidate for the management of NAFLD as it has been proven to be efficacious in the case of alcoholic liver hepatitis [46].

On the other hand, evidence correlating lycopene intake, either as a single carotene or as lycopene-containing foods, has so far failed to provide conclusive evidence of its protective efficacy on NAFLD [47]. This limitation is likely to be multifactorial, as assignable to the wide range of individual variability in the metabolism of lycopene into its bioavailable cis configuration [18] and by the need to resort to high consumption of lycopene-containing foods lacking defined nutrients profiles [48] and associated with high calories uptake. It should be underlined that lycopene with a daily requirement of 0,5 mg/kg and a plasma elimination half-life of 5 days [49,50], taken in the range of 5 to 7 mg/day [51] is mainly deriving from lycopene E 160d red food coloring agent intake [50].

To overcome these limitations, an improved formulation of this functional food has been reported using whole tomato fruits in view of their lipid metabolism modulating activity [28,52,53]. This WTFS, produced by calibrated heating of the berry and spray drying [28] is biofortified with olive wastewater micronutrients and by the presence of Fru-His Amadori’s chelators [54], displays an overall superior composition compared to available tomato commodities. WTFS is also endowed with the ability to interfere with metabolic pathways mediating oxidative stress and inflammation, as demonstrated by in vitro [33], animal [55] and human conditions of known susceptibility to tomato micronutrients benefits [29].

Within the limits of using a transformed hepatocyte cell line [56], the information gathered in this study may be informative to explore strategies to contrast liver lipid storage. Indeed, although the progression of NAFLD is the result of a stepwise engagement of other parenchymal, i.e .Kuppfer cells [57], stellate cells [58], and non-parenchymal, i.e. immune cells [59], the drivers of this progressive disease stem from the triglycerides accumulation [60] and lipogenesis [61] in hepatocytes overrunning their dismissal capacity through fatty acid oxidation and/or higher production rates of very-low-density lipoprotein particles [62].

Although the ability of WTFS single components to downmodulate the lipidomic asset of HepG2 cells cannot be fully appreciated, a converging ability of lycopene and other WTFS components tocopherol, tyrosol, hydroxytyrosol, oleopeurina, in lowering tryglycerides through also inhibition of STAT-3 phosphorylation [63], and AhR receptors activation [64] can be hypothesized [33].

Considering that WTFS has been shown to interfere with a several cell signaling, i.e. TRK receptor activation, nuclear factor-kappa B (NF-κB), mitogen-activated protein kinases (MAPK), upregulating the recruiting of inflammatory cells and inhibiting JAK/STAT kinases modulating inflammatory genes [33], the proteomic analysis identified three major targets of WTFS in HepG2 cells, which, by variably activating different signaling, may interfere with multiple metabolic pathways, thus modulating mechanisms fostering the progression of NAFLD. Indeed, a) TGF-α, by engaging the EGF receptor, triggers multiple downstream signaling, i.e. RTK, phosphatidylinositol-3 kinase (PI3K), ERK, and mTOR, which are relevant to liver regeneration [65], thus being a target of WTFS complex of micronutrients. In addition, TGF-α has been identified as an independent indicator of significant liver fibrosis [66]; b) TWEAK, a mitogen for liver progenitor cells [67], while undetectable in normal liver, it is significantly upregulated in patients with fatty liver than in other liver diseases, thus offering a potential therapeutic target [68], also in view that lowering its signaling in vivo may decrease levels of inflammation [36,69] and unbalanced signaling may lead to altered tissue remodeling [70].

Modelling of ligand and receptor interactions at multi cellular level [71] and integrative single-cell and spatial transcriptomic analyses have identified TNFRSF12A [72], as a relevant actor in supporting fibrinogenesis in liver pathology and a potential therapeutic target [73]; c) activation of FLT3LG, which through binding to the receptor Flt3/CD135, causes dimerization and autophosphorylation of the receptor with activation of iPI3K/Akt/mTOR, JAK/STAT, and RAS/RAF/Erk pathways [74], concurs to fibrosis [75] through epithelial mesenchymal transition [76]. In this regard, WTFS contains a complex of anti fibrogenic nutrients (lycopene, quercetin, narigenin, verbascoside) which can modulate epithelial-mesenchymal transition [77]. Our STRING network analysis revealed significant functional enrichment in key molecular functions among the input proteins, notably “cytokine activity” and “receptor ligand activity”. The enrichment of cytokine activity, as evidenced by the clustering of multiple nodes, suggests a prominent role for cytokine-mediated signaling pathways in the biological context under investigation. This aligns with the known involvement of cytokines in modulating inflammatory responses and cellular communication, particularly in pathological states such as immune activation or tissue remodeling. Moreover, the identification of receptor-ligand activity underscores the functional relevance of proteins capable of engaging receptor-mediated mechanisms, which are critical for transducing extracellular signals into specific cellular responses. Together, these enriched terms point toward a functional network characterized by intercellular communication and signal transduction, offering mechanistic insights into the observed phenotypic effects and highlighting potential targets for further experimental validation.

A constant dietary supplementation with WTFS containing a complex of highly bioactive nutrients with share biological activities with lycopene may have in vivo healthy effects that go beyond those produced on hepatocytes since it can modulate high-density lipoprotein [78], multiple signaling relevant to progression of NAFLD because upregulated in variety of cell types contributing to inflammation, angiogenesis, and fibrosis. It should be recalled that WTFS has been shown in fact to interfere with signaling of a number of cytokine and chemokines [55] and to block STAT-3 activation involved in NAFLD progression [79]. Furthermore, the WTFS content of highly bioavailable cis lycopene may be advantageous in patients with NAFLD whose liver has impaired ability to generate lycopene active metabolites (i.e. apo-lycopenals and apo-lycopenones) [10].

Although interventional studies will assess by what extent WTFS or similar supplements may ameliorate NAFLD [24], this functional food free of tomato culinary-associated side effects [30] is an attractive candidate to complement the Mediterranean diet, highly recommended for the prevention/treatment of NAFLD [80] but now recognized as hard to follow [81], thus hampering its wide compliance by large population fractions [82]. Those include patients with glucose intolerance [83], in whom NAFLD is often co-existing [84] but refrain from consuming high-calorie tomato-seasoned dishes, the main source of adequate amounts of bioavailable carotenoids. Since tomatoes are largely present in the international cuisine, the possibility of the culinary use of WTFS or similar products capable of fortifying meals may provide a lifetime efficacious strategy of delivering whole tomato improved nutrients.