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Introduction: Paternal nutrition before conception has a marked impact on offspring's risk of developing metabolic disorders during adulthood. Research on human cohorts and animal models has shown that paternal obesity alters sperm epigenetics (DNA methylation, protamine-to-histone replacement, and non-coding RNA content), leading to adverse health outcomes in the offspring. So far, the mechanistic events that translate paternal nutrition into sperm epigenetic changes remain unclear. High-fat diet (HFD)-driven paternal obesity increases gonadic Reactive Oxygen Species (ROS), which modulate enzymes involved in epigenetic modifications of DNA during spermatogenesis. Thus, the gonadic pool of ROS might be responsible for transducing paternal health status to the zygote through germ cells., Methods: The involvement of ROS in paternal intergenerational transmission was assessed by modulating the gonadic ROS content in male mice. Testicular oxidative stress induced by HFD was counterbalanced by N-acetylcysteine (NAC), an antioxidant precursor of GSH. The sires were divided into four feeding groups: i) control diet; ii) HFD; iii) control diet in the presence of NAC; and iv) HFD in the presence of NAC. After 8 weeks, males were mated with females that were fed a control diet. Antioxidant treatment was then evaluated in terms of preventing the HFD-induced transmission of dysmetabolic traits from obese fathers to their offspring. The offspring were weaned onto a regular control diet until week 16 and then underwent metabolic evaluation. The methylation status of the genomic region IGFII/H19 and cyp19a1 in the offspring gDNA was also assessed using Sanger sequencing and methylation-dependent qPCR., Results: Supplementation with NAC protected sires from HFD-induced weight gain, hyperinsulinemia, and glucose intolerance. NAC reduced oxidative stress in the gonads of obese fathers and improved sperm viability. However, NAC did not prevent the transmission of epigenetic modifications from father to offspring. Male offspring of HFD-fed fathers, regardless of NAC treatment, exhibited hyperinsulinemia, glucose intolerance, and hypoandrogenism. Additionally, they showed altered methylation at the epigenetically controlled loci IGFII/H19 and cy19a1., Conclusion: Although NAC supplementation improved the health status and sperm quality of HFD-fed male mice, it did not prevent the epigenetic transmission of metabolic disorders to their offspring. Different NAC dosages and antioxidants other than NAC might represent alternatives to stop the intergenerational transmission of paternal dysmetabolic traits., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Pastore, Badolati, Manfrevola, Sagliocchi, Laurenzi, Musto, Porreca, Murolo, Chioccarelli, Ciampaglia, Vellecco, Bucci, Dentice, Cobellis and Stornaiuolo.)

Wingless/integrase-11 (WNT)/β-catenin pathway is a crucial upstream regulator of a huge array of cellular functions. Its dysregulation is correlated to neoplastic cellular transition and cancer proliferation. Members of the Dishevelled (DVL) family of proteins play an important role in the transduction of WNT signaling by contacting its cognate receptor, Frizzled, via a shared PDZ domain. Thus, negative modulators of DVL1 are able to impair the binding to Frizzled receptors, turning off the aberrant activation of the WNT pathway and leading to anti-cancer activity. Through structure-based virtual screening studies, we identified racemic compound RS4690 ( 1 ), which showed a promising selective DVL1 binding inhibition with an EC 50 of 0.74 ± 0.08 μM. Molecular dynamic simulations suggested a different binding mode for the enantiomers. In the in vitro assays, enantiomer ( S )- 1 showed better inhibition of DVL1 with an EC 50 of 0.49 ± 0.11 μM compared to the ( R )-enantiomer. Compound ( S )- 1 inhibited the growth of HCT116 cells expressing wild-type APC with an EC 50 of 7.1 ± 0.6 μM and caused a high level of ROS production. These results highlight ( S )- 1 as a lead compound for the development of new therapeutic agents against WNT-dependent colon cancer.

Therapy-induced senescence (TIS) is a major cellular response to anticancer therapies. While induction of a persistent growth arrest would be a desirable outcome in cancer therapy, it has been shown that, unlike normal cells, cancer cells are able to evade the senescence cell cycle arrest and to resume proliferation, likely contributing to tumor relapse. Notably, cells that escape from TIS acquire a plastic, stem cell-like phenotype. The metabolic dependencies of cells that evade senescence have not been thoroughly studied. In this study, we show that glutamine depletion inhibits escape from TIS in all cell lines studied, and reduces the stem cell subpopulation. In line with a metabolic reliance on glutamine, escaped clones overexpress the glutamine transporter SLC1A5. We also demonstrate a central role of glutamine synthetase that mediates resistance to glutamine deprivation, conferring independence from exogenous glutamine. Finally, rescue experiments demonstrate that glutamine provides nitrogen for nucleotides biosynthesis in cells that escape from TIS, but also suggest a critical involvement of glutamine in other metabolic and non-metabolic pathways. On the whole, these results reveal a metabolic vulnerability of cancer stem cells that recover proliferation after exposure to anticancer therapies, which could be exploited to prevent tumor recurrence.

Phytochemical analysis of the Iranian plant Achillea wilhelmsii led to the isolation of 17 pure secondary metabolites belonging to the classes of sesquiterpenoids and phenolics. Two of these compounds, named wilhemsin ( 7 ) and wilhelmsolide ( 9 ), are new sesquiterpenoids, and the first shows undescribed structural features. Their structures were elucidated through extensive spectroscopic analysis, mainly based on 1D and 2D NMR, and chemical derivatization. Starting from plant traditional use and previous reports on the activity of the plant extracts, all the pure compounds were evaluated on endpoints related to the treatment of metabolic syndrome. The sesquiterpene hanphyllin ( 8 ) showed a selective cholesterol-lowering activity (-12.7% at 30 µM), santoflavone ( 13 ) stimulated glucose uptake via the GLUT transporter (+16.2% at 30 µM), while the trimethoxylated flavone salvigenin ( 14 ) showed a dual activity in decreasing lipid levels (-22.5% palmitic acid biosynthesis at 30 µM) and stimulating mitochondrial functionality (+15.4% at 30 µM). This study further confirms that, in addition to the antioxidants vitexin, isovitexin, and isoschaftoside, A. wilhelmsii extracts contain molecules that can act at different levels on the metabolic syndrome symptoms.

A large body of evidence suggests that supplementation of butyric acid exerts beneficial intestinal and extra-intestinal effects. Unfortunately, unpleasant sensorial properties and unfavourable physico-chemical properties strongly limit its use in food supplements and foods for medicinal purposes. N-(1-carbamoyl-2-phenyl-ethyl) butyramide (FBA) is a new butyric acid releaser in solid form with neutral sensorial properties. The aim of this investigation is to provide preliminary information on its pharmacokinetic and toxicological properties through the study of a) in vivo bioavailability of FBA administered by oral gavage to male and female Swiss CD1 mice in comparison with sodium butyrate, b) the influence of digestion on FBA stability through an in vitro simulated oro-gastro-duodenal digestion process, and c) in vitro toxicological profile by means of the Ames Test and Micronucleus Test. The results reveal that FBA is a good butyric acid releaser, being able to increase butyrate serum concentration in a dose and time dependent manner in both male and female mice with a pharmacokinetic profile similar to that obtained from sodium butyrate as such. These data are confirmed by investigating the influence of digestion on FBA, which undergoes extensive hydrolysis following oro-gastro-duodenal digestion, especially in duodenal conditions, with a residual concentration of less than 10% of the initial FBA concentration. Finally, in the Ames and Micronucleus Tests, FBA does not show any in vitro genotoxicity as it is non mutagenic in the Ames Test and results to be unable to induce chromosome breaks in the Micronucleus Test. In conclusion, FBA is a new butyric acid releaser that can overcome the disadvantages of butyric acid while maintaining the same pharmacokinetic properties and safety profile, as shown by the results of the preliminary in vitro toxicological studies performed in this investigation., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Grapes and apples are the most cultivated fruits in the Mediterranean basin and their agricultural processing is responsible for the production of a large amount of bio-waste. The reuse of this food biomass would increase the volume of recyclable/renewable biomaterial and lower the environmental impact due to the increasing demand for these biological products. To this purpose, agri-food waste from grape and apple processing have become an important source of phytochemicals, and many pharmaceutical industries are using it as starting material to produce dietary supplements, functional foods, and food additives for human consumption. In virtue of the chemical diversity and complexity of agri-food biowaste, developers and producers of nutraceuticals are advised to assess the safety of their final nutraceutical products, in compliance with European Food Safety Authority regulation. Here, we use the Ames test to assess the mutagenicity of three nutraceuticals obtained from agri-food waste biomasses: Taurisolo ® from grape pomace of Vitis vinifera L. cv 'Aglianico', AnnurComplex ® from Malus pumila M. cv 'Annurca' and Limoncella Apple Extract from Malus domestica B. cv 'Limoncella'. The results showed that all three nutraceuticals were non-mutagenic.

Liver diseases affect millions of people worldwide. In most of the cases, severe hepatic dysfunction and liver cancer stem from mild and common clinical signs including hepatic steatosis, insulin resistance, liver inflammation, and oxidative stress, all together referred to as Nonalcoholic Fatty Liver Disease (NAFLD). Nutraceuticals endowed with antioxidant activity have been shown to reduce NAFLD risk factors and exert hepatoprotective effects. Here, we test the protective effect exerted on liver by the antioxidant Taurisolo, a nutraceutical formulation produced by grape pomace and enriched in Resveratrol and Polyphenols. We analyze the effect of Taurisolo on liver cells by profiling the metabolome of in vitro cultured hepatic HuH7 cells and of C57BL-6J mice fed a High Fat Diet and treated with the nutraceutical. Both in vitro and in vivo, we provide evidence that Taurisolo reduces risk factor markers associated with NAFLD. Taurisolo stimulates glucose uptake and reduces hepatic cholesterol and serum triglycerides. Furthermore, we give new insights into the mechanism of action of Taurisolo. The nutraceutical increases mitochondrial activity and promotes respiration and ATP production, fostering catabolic reactions like fatty acid β-oxidation and amino acid catabolism. On the contrary, Taurisolo reduces anabolic reactions like biosynthesis of cholesterol, bile acids, and plasma membrane lipids., Competing Interests: The authors declare no conflict of interest.

Reduction in cholesterol blood levels represents one of the therapeutic goals to achieve in order to reduce the occurrence of cardiovascular diseases. Commonly, this goal is attempted by promoting healthy lifestyle behaviors and low-fat diets. Recently, several nutraceuticals have been shown to possess cholesterol-lowering properties and are becoming common over the counter products. Among others, apple polyphenols efficiently lower total cholesterol levels in humans and impact overall lipid metabolism. Malus Pumila Miller cv Annurca is an apple native to Southern Italy presenting one of the highest content of procyanidin B2, a dimeric procyanidin. Tested in clinical trials, the oral consumption of an Annurca polyphenolic extract (AAE) exerted a cholesterol-lowering effect similar to the statins Atorvastatin and Simvastatin. Despite AAE activity, the analysis of the molecular mechanism behind its cholesterol-lowering effect is unclear. Using isotope labeling and high-resolution mass spectrometry approaches we here performed a metabolic profiling of in vitro cultured human hepatocytes treated with AAE to reveal its mechanism of action. The results show that AAE acts differently than statins. The extract reprograms hepatic cell metabolism and promotes mitochondrial respiration, lipolysis and fatty acid β-oxidation. Citrate and acetyl-CoA, both necessary for the production of cholesterol, are diverted to the Krebs Cycle by AAE, that, ultimately, lowers cholesterogenesis and fatty acid synthesis.

Chemotherapy-induced alopecia (CIA) is a common side effect of conventional chemotherapy and represents a major problem in clinical oncology. Even months after the end of chemotherapy, many cancer patients complain of hair loss, a condition that is psychologically difficult to manage. CIA disturbs social and sexual interactions and causes anxiety and depression. Synthetic drugs protecting from CIA and endowed with hair growth stimulatory properties are prescribed with caution by oncologists. Hormones, growth factors, morphogens could unwontedly protect tumour cells or induce cancer cell proliferation and are thus considered incompatible with many chemotherapy regimens. Nutraceuticals, on the contrary, have been shown to be safe and effective treatment options for hair loss. We here show that polyphenols from Malus Pumila Miller cv Annurca are endowed with hair growth promoting activity and can be considered a safe alternative to avoid CIA. In vitro, Annurca Apple Polyphenolic Extract (AAE) protects murine Hair Follicles (HF) from taxanes induced dystrophy. Moreover, in virtue of its mechanism of action, AAE is herein proven to be compatible with chemotherapy regimens. AAE forces HFs to produce ATP using mitochondrial β-oxidation, reducing Pentose Phosphate Pathway (PPP) rate and nucleotides production. As consequence, DNA replication and mitosis are not stimulated, while a pool of free amino acids usually involved in catabolic reactions are spared for keratin production. Moreover, measuring the effect exerted on Poly Unsaturated Fatty Acid (PUFA) metabolism, we prove that AAE promotes hair-growth by increasing the intracellular levels of Prostaglandins F2α (PGF2α) and by hijacking PUFA catabolites toward β-oxidation.

Patterned hair loss (PHL) affects around 50% of the adult population worldwide. The negative impact that this condition exerts on people's life quality has boosted the appearance of over-the-counter products endowed with hair-promoting activity. Nutraceuticals enriched in polyphenols have been recently shown to promote hair growth and counteract PHL. Malus pumila Miller cv. Annurca is an apple native to Southern Italy presenting one of the highest contents of Procyanidin B2. We have recently shown that oral consumption of Annurca polyphenolic extracts (AAE) stimulates hair growth, hair number, hair weight and keratin content in healthy human subjects. Despite its activity, the analysis of the molecular mechanism behind its hair promoting effect is still partially unclear. In this work we performed an unprecedented metabolite analysis of hair follicles (HFs) in mice topically treated with AAE. The metabolomic profile, based on a high-resolution mass spectrometry approach, revealed that AAE re-programs murine HF metabolism. AAE acts by inhibiting several NADPH dependent reactions. Glutaminolysis, pentose phosphate pathway, glutathione, citrulline and nucleotide synthesis are all halted in vivo by the treatment of HFs with AAE. On the contrary, mitochondrial respiration, β-oxidation and keratin production are stimulated by the treatment with AAE. The metabolic shift induced by AAE spares amino acids from being oxidized, ultimately keeping them available for keratin biosynthesis., Competing Interests: The authors declare no conflict of interest.

The WNT pathway interconnects a network of signaling events involved in a huge plethora of cellular processes, from organogenesis to tissue homeostasis. Despite its importance, the exiguity of organic drugs directly targeting the members of the Frizzled family of WNT receptors has hampered progress across the whole spectrum of biological fields in which the signaling is involved. We here present FzM1.8, a small molecule acting as an allosteric agonist of Frizzled receptor FZD4. FzM1.8 derives from FzM1, a negative allosteric modulator of the receptor. Replacement of FzM1 thiophene with a carboxylic moiety induces a molecular switch in the lead and transforms the molecule into an activator of WNT signaling. We here show that, in the absence of any WNT ligand, FzM1.8 binds to FZD4, promotes recruitment of heterotrimeric G proteins, and biases WNT signaling toward a noncanonical route that involves PI3K. Finally, in colon cancer cells, we prove that the FZD4/PI3K axis elicited by FzM1.8 preserves stemness and promotes proliferation of undifferentiated cells.

Inhibitors of the Wingless-related Integration site (WNT)/β-catenin pathway have recently been under consideration as potential chemopreventive agents against Familial Adenomatous Polyposis (FAP). This autosomal-dominant syndrome is caused by germline mutations in the gene coding for the protein APC and leads to hyperactivation of the WNT/β-catenin signaling pathway, uncontrolled intestinal cell proliferation and formation of adenocarcinomas. The aim of the present work was to: (i) test, on in vitro cultures of cells carrying FAP mutations and on ex vivo biopsies of FAP patients, the WNT inhibitory activity of extracts from two common southern Italian apples, Malus pumila Miller cv. 'Annurca' and Malus domestica cv 'Limoncella'; (ii) identify the mechanisms underpinning their activities and; (iii) evaluate their potency upon gastrointestinal digestion. We here show that both Annurca and Limoncella apple extracts act as WNT inhibitors, mostly thanks to their polyphenolic contents. They inhibit the pathway in colon cells carrying FAP mutations with active dilutions falling in ranges close to consumer-relevant concentrations. Food-grade manufacturing of apple extracts increases their WNT inhibitory activity as result of the conversion of quercetin glycosides into the aglycone quercetin, a potent WNT inhibitor absent in the fresh fruit extract. However, in vitro simulated gastrointestinal digestion severely affected WNT inhibitory activity of apple extracts, as result of a loss of polyphenols. In conclusion, our results show that apple extracts inhibit the WNT pathway in colon cells carrying FAP mutations and represent a potential nutraceutical alternative for the treatment of this pathology. Enteric coating is advisable to preserve the activity of the extracts in the colon-rectal section of the digestive tract., Competing Interests: The authors declare no conflict of interest.