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Abstract

The lipidome of royal jelly (RJ) consists of medium-chained (8–12 carbon atoms) free fatty acids. We present herein a liquid chromatography-high resolution mass spectrometry (HRMS) method that permits the determination of RJ fatty acids and at the same time the detection of suspect fatty acids. The method allows for the direct quantification of seven free fatty acids of RJ, avoiding any derivatization step. It was validated and applied in seven RJ samples, where the major RJ fatty acid trans-10-hydroxy-2-decenoic acid (10-HDA) was found to vary from 0.771 ± 0.08 to 0.928 ± 0.04 g/100 g fresh RJ. Four additional suspect fatty acids were simultaneously detected taking advantage of the HRMS detection.

2.4. Analysis of RJ Samples

Seven different RJ samples, which were purchased from the local market, were analyzed. The extracted ion chromatograms (EICs) of the analytes in a standard solution (A), as well as in a representative RJ sample (B) are presented in . The isobaric 10-hydroxydecanoic acid and 3-hydroxydecanoic acid are well separated and are eluted at 2.37 and 3.25 min, respectively.

2.4. Analysis of RJ Samples

The concentrations of the seven analytes in seven RJ extracts (in triplicate) are summarized in . represents the multi sample analysis data. The contents of free fatty acids are expressed as g of fatty acid per 100 g of fresh RJ. 10-HDA was found to be the most abundant, as expected, and its content ranged from 0.771 ± 0.08 to 0.928 ± 0.04 g/100 g fresh RJ. These results are in accordance with literature taking into account that the content of 10-HDA depends on the origin of the jelly and the characteristics of the bee. The levels of 10-HDA have been found to vary from 0.75 to 2.54 by Genc and Aslan using column liquid chromatography [], and from 0.8 to 3.2 by Ferioli et al. using either CZE or HPLC [].

9-(S)-HODE modulates inflammatory responses

We next examined whether reduction of MPO activity could influence the severity of inflammatory responses in flies of the hopTum inflammation model raised on LA-supplemented media. Genetic reduction in MPO levels using the transposon mutant alleles Pxtf05258 and PxtEY03052 decreased tumour incidence (). This was replicated by pharmacological blockade of MPO activity by MPO inhibitor-1 treatment, which caused equivalent decreases in tumour incidence (). However, when media was supplemented with 9-(S)-HODE (the product of MPO oxidation of LA), genetic reduction in MPO levels using Pxtf05258 mutants failed to decrease tumour incidence (). Taken together, these results were consistent with MPO-mediated oxidation of the omega-6 PUFA LA generating 9-(S)-HODE that in turn modulates inflammatory responses.

Abstract

Background: Gut microbiota plays a pivotal role in regulating host metabolism that affects the systemic health. To date, several studies have confirmed the fact that microbiota interacts with host, modulating immunity, controlling the homeostasis environment, and maintaining systemic condition. Recent studies have focused on the protective function of poly unsaturated fatty acids, 10-oxo-trans-11-oxadecenoic acid (KetoC) and 10-hydroxy-cis-12-octadecenoic acid (HYA), generated by gut microbiota on periodontal disease. Nevertheless, the mechanism remains unclear as investigations are limited to in vivo and in vitro studies. In this present review, we found that the administration of metabolites, KetoC and HYA, by a probiotic gut microbiota Lactobacillus plantarum from linoleic acid is found to inhibit the oxidation process, possess an antimicrobial function, and prevent the inflammation. These findings suggest the promising use of functional lipids for human health. Conclusion: Protective modalities of bioactive metabolites may support periodontal therapy by suppressing bacterial dysbiosis and regulating periodontal homeostasis in the clinical setting.

2.2.1. Anti-Inflammatory Function

Regarding the signaling pathway regulating the inflammation, HYA is found to decrease TNFR2 expression in Dextran sulfate sodium (DSS)-induced colitis mice []. In respect to this, the upregulation of TNFR2 subsequently reduced the ratio of phospho-IκBα/IκBα and NFκB p65, thereby decreasing proinflammatory cytokine production. HYA decreases local inflammatory cytokine mRNA level (IL-1β, TNFα, and IL-6) in gingival tissue in vivo with a tendency of alveolar bone loss suppression []. HYA induces ERK phosphorylation; on the other hand, the blockade of the MEK-ERK signaling deteriorates the HYA-GPR40 anti-inflammatory action [].

2.2.3. Effect on Epithelial Barrier Function

HYA treatment suppressed this degradation by promoting the proteolytic resistance of E-cadherin/β-catenin toward P. gingivalis in the gingival epithelial cells [] and colorectal epithelial adenocarcinoma cells (Caco-2) [], thereby reducing the inflammation and injury. An investigation reported that tight junction permeability (TER) and protein molecules (occluding, ZO-1, ZO-2, and claudin-3) were impaired by INF-γ and TNFα challenge in Caco-2 cells through the upregulation of IL-8, and then recovered by the pretreatment of HYA in a dose-dependent manner []. Moreover, the action of HYA depends on the expression of GPR40 in gingival epithelial cells [].

Abstract

Resistance to antibiotics has made diseases that previously healed easily become more difficult to treat. Staphylococcus aureus is an important cause of hospital-acquired infections and multi-drug resistant. NorA efflux pump, present in bacteria S. aureus, is synthesized by the expression of the norA gene. Menadione, also known as vitamin K3, is one of the synthetic forms of vitamin K. Therefore, the aim of this study is to verify the menadione effect on efflux inhibition through NorA pump gene expression inhibition and assess the effects of menadione in bacterial membrane. The effect of menadione as an efflux pump inhibitor (EPI) was evaluated by the microdilution method, fluorimetry, electron microscopy, and by RT-qPCR to evaluate gene expression. In the molecular docking, association with menadione induces increased fluorescence intensity. Menadione was observed (100% of the clusters) interacting with residues ILE12, ILE15, PHE16, ILE19, PHE47, GLN51, ALA105, and MET109 from NorA. The results showed the norA gene had its expression significantly diminished in the presence of menadione. The simulation showed that several menadione molecules were able to go through the bilayer and allow the entry of water molecules into the hydrophobic regions of the bilayer. When present within membranes, menadione may have caused membrane structural changes resulting in a decline of the signaling pathways involved in norA expression. Menadione demonstrated to be an efflux pump inhibitor with dual mechanism: affecting the efflux pump by direct interaction with protein NorA and indirectly inhibiting the norA gene expression, possibly by affecting regulators present in the membrane altered by menadione.

2.1. Bacterial Strain and Culture

The SA-1199B strain of Staphylococcus aureus was kindly donated by Prof. S. Gibbons of the University of London. Before all microbiological tests, the strain was maintained in the culture medium blood agar base (Laboratorios Difco Ltd., São Paulo, Brazil). For use in testing, the cells were grown overnight at 37 °C in the culture medium: Heart Infusion Agar (HIA, Difco). The strain used has the generic characteristic of presenting the norA resistance gene responsible for expressing the efflux pump (NorA) for antibiotics in the group of fluoroquinolones.

2.7. Sample Preparation for the Real-Time PCR Assay

The samples were prepared using the minimum inhibitory concentration (MIC) as obtained in Tintino et al. []. The assays were performed as described in the . The preparations were cultured in Brain Heart Infusion (BHI) culture medium for 24 h at 37 °C and then centrifuged at 3000 rpm (Fanem Centrífuga Excelsa® 3280) for pellet formation, procedures performed prior to RNA extraction.

3.1. Efflux Pump Inhibition by MIC Reduction of Ethidium Bromide (EtBr)

In the evaluation of inhibition by MIC reduction of ethidium bromide, the results in , menadione was shown to significantly reduce the ethidium bromide MIC, which indicates NorA inhibition. In the inhibition of the efflux pump, it is known that decreasing the MIC of bromide, of ethidium or antibiotic, by up to three-fold, in relation to the control (substrate of efflux pump) alone, is indicative of efflux pump inhibition.

3.2. Evaluation of Pump Inhibition by Fluorescence Emission of Ethidium Bromide

In the evaluation of inhibition by fluorescence emission of ethidium bromide, according to the results in , the association with menadione induces increased fluorescence intensity when compared to the bacterium associated only with bromide, indicating inhibition of efflux pump. Regarding the Carbonyl Cyanide m-Chlorophenylhydrazine (CCCP) pump inhibitor, the same showed low fluorescence intensity when compared to the others.

3.3. Homology Modeling and Molecular Docking

The molecular docking method was used to investigate the binding affinity of menadione in the channel formed by the transmembrane alpha-helices of the NorA protein. Menadione was observed (100% of the clusters) interacting with residues ILE12, ILE15, PHE16, ILE19, PHE47, GLN51, ALA105, and MET109 from NorA (). This protein region has also been observed in other docking studies in the literature with other binders such as boeravinone B [], riparin B [], salvin, thioxanthene, and levofloxacin [].

Abstract

Vitamin D and its analogs are known for their role in the development of breast cancer and in immunomodulation. Our previous studies have shown the pro-metastatic effect of calcitriol and tacalcitol (PRI-2191) in young mice bearing 4T1 breast cancer and the anti-metastatic effect in aged ovariectomized (OVX) mice. Therefore, the aim of our work was to characterize Th17 cell population in young and aged OVX mice bearing 4T1 tumors treated with calcitriol and PRI-2191. The expression of genes typical for Th17 cells was examined in splenocytes, as well as splenocytes differentiated with IL-6 and TGF-β to Th17 cells (iTh17). Expression of genes encoding vitamin D receptor (Vdr) and osteopontin (Spp1) as well as the secretion of IL-17A were evaluated in iTh17 cells. PRI-2191 treatment increased the expression of Rora and Rorc transcription factors, Il17a, Il17re and Il21 in iTh17 cells from young mice. In aged OVX mice this effect was not observed. Increased expression was observed in the case of Vdr and Spp1 genes in iTh17 cells from young mice treated with PRI-2191. What is more, in young mice treated with PRI-2191 the secretion of IL-17A to the culture media by iTh17 cells was increased, whereas in aged OVX mice a significant decrease was noted. Increased expression of Spp1 in young mice treated with PRI-2191 may enhance the differentiation of Th17 cells.

Splenocytes and lymph nodes used in the studies

The splenocytes and lymph nodes utilized in this study were collected from female BALB/c mice used in experiments described in our previous work [,]. The course of experiment on 6-8-week-old mice weighing 20-25 g was described in [] and on 60-week-old mice weighing 24-30 g in [] (mice obtained from the Center of Experimental Medicine of the Medical University of Bialystok, Poland). All methods used in those experiments were compliant with the EU Directive 2010/63/EU on the protection of animals used for scientific purposes and was approved by the First Local Committee for Experiments with the Use of Laboratory Animals, Wroclaw, Poland (no. of permission: 40/2014). Calcitriol (1,25(OH)2D3) and PRI-2191 (1,24(OH)2D3, tacalcitol) were obtained from the Pharmaceutical Research Institute, Warsaw, Poland. PRI-2191 was described as compound with lowered calcemic activity and increased anticancer activity as compared to calcitriol [,].

Splenocytes and lymph nodes used in the studies

Briefly, 1×104 4T1 mammary gland cancer cells (American Type Culture Collection, Rockville, MD, USA) per mouse were injected orthotopically into young mice [] - as a premenopausal model or into old mice four weeks after they had been ovariectomized [] - as a postmenopausal model. Starting from day 7 after cells inoculation, mice were subcutaneously (s.c.) injected with 80% propylene glycol (vehicle), calcitriol (0.5 µg/kg) and PRI-2191 (1.0 µg/kg) three times a week. On days 0, 7, 14, 21, 28, and 33 after transplantation, spleens and lymph nodes were harvested after euthanasia of mice.

Splenocytes and lymph nodes used in the studies

The experiment on young mice was repeated twice, whereas the experiment on aged OVX mice was conducted once.

PRI-2191 increased the Th17 genes expression in iTh17 splenocytes from young mice bearing 4T1 mouse mammary gland cancer

CD4+ splenocytes stimulated by IL-6 and TGF-β to Th17 cells had different expression level of selected genes when harvested from young and old OVX mice. The induced iTh17 cells from splenocytes of young mice treated with PRI-2191 expressed significantly higher levels of Il17a mRNA as compared to iTh17 splenocytes from control tumor bearing mice (). The expression of transcription factors Rora and Rorc and IL-17 receptor (Il17re) as well as IL-21 (Il21) mRNA was higher in iTh17 cells from mice treated with PRI-2191 (). Vitamin D receptor mRNA (Vdr) as well as osteopontin (Spp1) significantly increased in iTh17 cells from young mice treated with PRI-2191 (). Calcitriol did not affect in statistically significant manner the expression of genes in young mice (). Aged OVX mice did not reveal any significant changes in the expression levels of analyzed genes (). The purity of isolated CD4+ splenocytes from young () and aged OVX () were controlled by flow cytometry after staining of isolated cells with anti-CD4 antibody.

Abstract

Retinyl palmitate is a vitamin A ester belonging to the family of endogenous natural retinoid and used to treat various skin disorders like acne, skin aging, wrinkles, and dark spots, as well as to protect against psoriasis. Despite the known therapeutic benefits of retinyl palmitate, the conventional topical delivery of retinyl palmitate commonly associated with adverse reactions such as skin irritation, redness, excessive peeling, and dryness. Therefore, the current study aims to encapsulate the retinyl palmitate in nanoemulsion then incorporate it into a hydrogel system to improve the topical delivery and stability. Low-energy emulsification method was used for the nano-encapsulation of retinyl palmitate. The phase behavior study was used for the investigation and the optimization of the formulation. The droplet size of the optimized nanoemulsion was in nano dimension (16.71 nm) with low polydispersity index (PdI) (0.015), negative zeta potential (−20.6 mV). It demonstrated the influence of vortexing on droplet size and PdI during nanoemulsion preparation. The retinyl palmitate loaded nanoemulgel delivery system exhibited significant improvement (p < 0.05) in skin permeability after topical application. Employment of the nano-encapsulation approach afterward dispersion into hydrogel system for the development of a topical delivery system of retinyl palmitate resulted in improvement in its UV and storage stability as well.

Abstract

In plants, metabolic homeostasis—the driving force of growth and development—is achieved through the dynamic behavior of a network of enzymes, many of which depend on coenzymes for activity. The circadian clock is established to influence coordination of supply and demand of metabolites. Metabolic oscillations independent of the circadian clock, particularly at the subcellular level is unexplored. Here, we reveal a metabolic rhythm of the essential coenzyme thiamine diphosphate (TDP) in the Arabidopsis nucleus. We show there is temporal separation of the clock control of cellular biosynthesis and transport of TDP at the transcriptional level. Taking advantage of the sole reported riboswitch metabolite sensor in plants, we show that TDP oscillates in the nucleus. This oscillation is a function of a light-dark cycle and is independent of circadian clock control. The findings are important to understand plant fitness in terms of metabolite rhythms.

Introduction

Here, in addition to demonstrating the importance of TDP for central metabolism and thereby growth and development, we report on components of the intracellular transport of TDP and/or its precursors in the plant model Arabidopsis. We show that both TDP biosynthesis and transport are transcriptionally regulated by the circadian clock. Importantly, biosynthesis is temporally separated from transport and is likely an important parameter for coordination of TDP supply and demand. We have used the alternative splicing of the THIC riboswitch as a natural sensing tool to report on daily modulation of TDP levels. Indeed, levels of TDP oscillate at the intracellular level (i.e. the nucleus) peaking in abundance in the first half of the day. This peak in abundance is derivative of light-dark cycles that is independent of the circadian clock regulation.

TDP is essential for central metabolism

During seed development/maturation in plants, the nonphosphorylated B1 vitamer thiamine is stored, and upon germination is converted to TDP (Fig. ), furnishing enzymes of central metabolism in the cotyledons. Beyond this however, the development of true leaves requires biosynthesis de novo of TDP. This is corroborated by the fact that thiamine biosynthesis de novo mutants, such as thiC, cannot proceed beyond the cotyledon stage of development. The necessity of having biosynthesis de novo can be bypassed by supplementation with thiamine. Bypassing biosynthesis de novo in these previous experiments was performed by supplementing with excessive levels of thiamine (up to 100 μM). In order to test whether core metabolism is indeed dependent on TDP levels, we grew the thiC mutant in the presence of a range of thiamine concentrations (0−1.5 μM), spanning physiologically measured levels. Supplementation with thiamine had little effect on wild-type growth (Fig. ). However, the growth of thiC beyond the cotyledon stage was dependent on the level of thiamine supplementation (Fig. ). Moreover, TDP levels in these seedlings showed a clear correlation with thiamine supplementation levels (Fig. ). Furthermore, we utilized a nontargeted metabolomics approach to assess metabolite changes as a function of the thiamine supplementation level. Mapping the set of detectable metabolites onto a scheme of central metabolism illustrates the panorama of metabolite changes with and without thiamine supplementation in thiC (Supplementary Fig. ). Such changes are not seen in the corresponding wild type under the same conditions (Supplementary Fig. ). Across the range of metabolites measured, a core set of metabolites was seen to be significantly dependent on the level of thiamine supplementation (Fig. ). Notably, many of these metabolites were photosynthetic pigments. From the measurements of TDP levels in these seedlings, it appears that ca. 1 μmol g−1 FW (at the tissue level) is required for growth to approach wild-type levels (Fig. ), because stunting and chlorosis can still be observed with lower supplementation levels (Fig. ). These data therefore highlight the fact that TDP is needed in relatively small amounts but it is also evident that even a small deficit (low nM) severely reduces growth, likely by directly impacting photosynthesis, essentially halting development. The data thus demonstrate that TDP supply is essential for central metabolism and limitation of this coenzyme has major impacts on growth and development.

Abstract

The genus Nicotiana (Solanaceae) includes over 70 species, with a long history of traditional use; many of them are nowadays used in bioengineering, biosynthesis, molecular biology, and other studies, while common tobacco, N. tabacum L., is one of the most economically important industrial crops worldwide. Although Nicotiana species have been extensively investigated, relatively less research has focused on flowers, especially research related to obtaining aromatic products for cosmetic and perfumery use. On the other hand, there is evidence that Nicotiana flowers accumulate various secondary metabolites with a distinct aroma and biological activities, and the flowers represent a biomass available in sufficient quantities. Therefore, this study aimed to determinate the chemical composition (by GC-MS) and the olfactory profiles of a specific type of natural aromatic product (concrete), obtained from the flowers of four Nicotiana species, in a direct comparison between them. The yields of extracted concrete were sufficiently high, varying between the species, 1.4% (N. rustica L.), 2.5% (N. glutinosa L.), 1.6% (N. alata Link&Otto genotype with white flowers), 2.7% (N. alata genotype with pink flowers), 3.2% (N. tabacum, Oriental type), and 5.2% (N. tabacum, Virginia type). The major components of the obtained concretes belonged to different chemical classes: N. rustica and N. tabacum (OR), the hydrocarbons n-tetratriacontane (14.5%; 15.0%) and n-triacontane (12.1%; 13.3%), and 3-methyl-pentanoic acid (11.1%; 12.2%); N. glutinosa, the diterpenes sclareol (25.9%), 3-α-hydroxy-manool (16.3%), and 13-epimanool (14.9%); N. alata (WF), the phenylpropanoid terephthalic acid and di(2-ethylhexyl) ester (42.9%); N. alata (PF), the diterpene tributyl acetylcitrate (30.7%); and N. tabacum (FCV), the hydrocarbons n-hexacosane (12.9%) and n-pentacosane (12.9%). Each of the flower concretes revealed a characteristic odor profile. This is the first report about Nicotiana species as a source for obtaining flower concretes; these initial results about the concrete yield, olfactory profile, and chemical composition are a prerequisite for the possible processing of Nicotiana flowers into new aromatic products for use in perfumery and cosmetics. The study provides new data in favor of the potential of the four Nicotiana species as aromatic plants, as well as a possible alternative use of flowers, a valuable, but discarded, plant material in other applications.

2. Results

In the concrete obtained from N. rustica flowers, 24 components (99.5% of the total content) were identified; only one of them had a concentration under 1% As seen from , the major constituents (over 3%) of the extract were: n-tetratriacontane (14.6%), n-triacontane (12.1%), pentanoic acid, 3-methyl (11.1%), tributyl acetylcitrate (9.1%), n-octacosane (5.5%), n-nonacosane (4.4%), n-dotriacontane (4.3%), n-hexatriacontane (3.7%), and (Z,Z)-linoleic acid (3.4%). The dominant group of substances in the concrete was that of aliphatic hydrocarbons (65.2%), and the rest of the identified components represented oxygenated aliphatics (29.4%), alkaloids (2.9%), and phenyl propanoids (2.4%).

Abstract

Honeybees produce royal jelly (RJ) from their cephalic glands. Royal jelly is a source of nutrition for the queen honey bee throughout its lifespan and is also involved in fertility and longevity. Royal jelly has long been considered beneficial to human health. We recently observed that RJ delayed impairment of motor function during aging, affecting muscle fiber size. However, how RJ affects skeletal muscle metabolism and the functional component of RJ is as of yet unidentified. We demonstrate that feeding mice with RJ daily prevents a decrease in myofiber size following denervation without affecting total muscle weight. RJ did not affect atrophy-related genes but stimulated the expression of myogenesis-related genes, including IGF-1 and IGF receptor. Trans-10-hydroxy-2-decenoic acid (10H2DA) and 10-hydroxydecanoic acid (10HDAA), two major fatty acids contained in RJ. After ingestion, 10H2DA and 10HDAA are metabolized into 2-decenedioic acid (2DA) and sebacic acid (SA) respectively. We found that 10H2DA, 10HDAA, 2DA, and SA all regulated myogenesis of C2C12 cells, murine myoblast cells. These novel findings may be useful for potential preventative and therapeutic applications for muscle atrophy disease included in Sarcopenia, an age-related decline in skeletal muscle mass and strength.

3.1. pRJ Attenuates Denervation-Induced Skeletal Muscle Atrophy

To examine the effect of pRJ on skeletal muscle atrophy, C57BL/6 mice were fed on control or pRJ diets for four weeks, and then muscle atrophy was induced by denervation. Daily feeding of pRJ for 1 month had no significant effect on total body weight (A) or loss of total tibialis anterior muscle weight induced by denervation (B). However, pRJ prevented the decrease in skeletal muscle fiber diameter following denervation (C,D). In order to determine the mechanism by which pRJ prevents the decrease in muscle fiber size, we next compared the expression levels of atrophy, proliferation, or skeletal muscle differentiation genes. qPCR and Western blotting analysis showed that pRJ did not alter the expression of catabolic genes such as Atrogin-1 (A and 2K), Muscle ring finger protein 1 (MuRF1) (B), or Forkhead box O-1 (Foxo-1) (C). However, pRJ increased the expression of proliferation and differentiation-related genes such as Cyclin E1 (D), Cyclin A2 (E), or Myogenin (G). Protease-treated RJ had no significant effect on the upregulation of Mych1 (H), Myhc2 (), Myhc4 (), or Myhc7 (). Protease-treated RJ stimulated the upregulation of IGF-1 (I) and IGF receptor (IGFR) (J) and phosphorylation of AMPK (K).

3.2. pRJ Stimulates Myoblast Proliferation

Protease-treated royal jelly treatment for 48 hours increased the number of cells (A,B) as well as the expression of cell-cycle-related genes (C–F). Furthermore, Ki67 immunostaining showed that pRJ treatment increased the number of Ki67-positive proliferative cells (G,H). Trans-10-hydroxy-2-decenoic acid and 10-hydroxydecanoic acid are two fatty acids specifically occurring in RJ. Trans-10-hydroxy-2-decenoic acid and 10-hydroxydecanoic acid can be metabolized into 2DA and SA, respectively []. To determine whether RJ-derived fatty acids and/or their metabolic products affect proliferation, C2C12 cells were treated with the indicated compounds for 24, 48, and 72 hours, after which cell numbers were quantified by WST-8. As shown in A, 10H2DA, 10HDAA, 2DA, and SA all significantly increased cell proliferation (A). These RJ-related fatty acids also increased the protein levels of cell cycle genes such as Cyclin D1 and Cyclin A2 (B).

3.3. pRJ Stimulates Myoblast Differentiation

Finally, we examined the effect of pRJ and RJ-related fatty acid products on myoblast differentiation. C2C12 cells were induced to differentiate in the presence or absence of pRJ. Myosin heavy-chain immunostaining showed that pRJ treatment led to an increase in myotube formation compared to control treatment cells (A,B). Furthermore, pRJ treatment elevated the expression level of muscle differentiation genes such as MyoD, Myogenin, Mck, or Myhc1 (C–H). C3H10T1/2 cell model is a mouse embryonic fibroblast cell line with myogenic potential. C3H10T1/2 cells, however, do not normally express MyoD, a master regulator of myogenesis []. To evaluate the effect of pRJ on MyoD function, we overexpressed MyoD in C3H10T1/2 cells and then treated the cells with or without pRJ. qPCR analysis revealed that pRJ treatment enhanced the induction of Myogenin and Myhc1 induced by MyoD (I,J). Treatment with 10H2DA, 10HDAA, 2DA, and SA increased myotube formation (A,B). The treatment of cells with these RJ-related fatty acids increased the protein levels of myogenic differentiation marker genes such as Myhc and myogenin (C).

4. Discussion

In this study, we demonstrate that feeding mice daily with pRJ prevents a decrease in myofiber size following denervation. In a previous study, we showed that pRJ affects muscle fiber size in elderly mice without changing total muscle weight []. Our current findings did not conflict with this study. Muscle weight during pathological conditions such as obesity, type 2 diabetes, or age-related Sarcopenia can be affected by the infiltration of adipose and/or connective tissue []. In our study, we did not quantify the infiltration of adipose or connective tissue into the muscle tissue, even though pRJ treatment tended to increase muscle weight. It may be interesting to explore the effect of RJ on the infiltration of muscle by adipose and connective tissue. Our data showed that daily feeding with pRJ did not decrease the expression levels of atrophy-related genes such as Atrogin-1, Murf1, or Foxo-1 although our previous work showed that pRJ feeding decreases atrophy-related gene expression during aging []. This discrepancy might be explained by differences in the relative extent of atrophy induced by denervation in comparison to aging. Nevertheless, differences between the two experimental models may be helpful to clarify the effect of RJ on skeletal muscle metabolism.

4. Discussion

We observed that RJ treatment stimulated both proliferation and differentiation. RJ stimulates cell proliferation and increases the size of Myhc positive fibers in primary satellite cells isolated from aged mice via upregulation of IGF-1 and IGFR []. In this study, RJ treatment significantly increased expression of IGF-1 and IGFR, suggesting that the IGF1-Akt pathway may contribute to the phenotype. In in vivo experiments, RJ treatment did not increase the expression levels of cyclin D1, whereas RJ treatment in vitro strongly increased cyclinD1. Currently, it is difficult to explain this discrepancy. Furthermore, we could not reveal which cell types are proliferating and differentiating in vivo following RJ treatment. This will be an important issue to resolve in future studies.

Abstract

The king scallop Pecten maximus retains the amnesic shellfish poisoning toxin, domoic acid (DA), for a long time. Most of the toxin is accumulated in the digestive gland, but this organ contains several cell types whose contribution to the accumulation of the toxin is unknown. Determining the time-course of the depuration by analyzing whole organs is difficult because the inter-individual variability is high. A sampling method, using biopsies of the digestive gland, has been developed. This method allows for repetitive sampling of the same scallop, but the representativeness of the samples obtained in this way needs to be validated. In this work, we found that the distribution of DA in the digestive gland of the scallops is mostly homogeneous. Only the area closest to the gonad, and especially its outer portion, had a lower concentration than the other ones, probably due to a transfer of the toxin to the intestinal loop. Samples obtained by biopsies can therefore be considered to be representative. Most of the toxin was accumulated in large cells (mostly digestive cells), which could be due to differences during the toxin absorption or to the preferential depuration of the toxin from the small cells (mostly secretory).

2.2. Distribution of Domoic Acid between Cellular Types

Domoic acid was preferentially accumulated into large (mostly digestive) cells (A, p = 0.0039 by Wilcoxon paired samples test). The measured concentration of this toxin in small cells (basophilic, mostly secretory) was less than 25% of that in large cells.

3. Discussion

The variability of the toxin concentration among areas of the digestive gland of the scallops is moderate-low. It is lower than the usual inter-individual variation in scallops [,] and other bivalves [,,]. Most sectors of the digestive gland had very similar concentrations, with the only significant exception being the sector adjacent to the gonad. In addition, in this sector the outer region was shown to have a lower concentration than the inner one. The most likely cause for this difference is the migration of the toxin to the intestinal loop located in the gonad. In previous studies [], it has been observed that gonadal growth during depuration produces an increase in DA burden in this organ. The origin of the toxin incorporated by the intestinal loop should be the digestive gland, which is where the DA in the scallops is mainly stored. The mechanism involved, nevertheless, is unknown. A possibility could be the reabsorption of the toxin excreted by the digestive gland by the intestinal cells. It has been shown that those cells can significantly absorb different substances [], but the very low depuration rate of the scallops [,] suggests that the amount of DA that could be reabsorbed is very low. In any case, this mechanism would not be expected to affect some parts of the gonad differently. A second possibility could be the transfer of the toxin from the digestive gland cells to the newly generated intestinal ones. Transfers of DA between organs have been documented but are significant only in exceptional situations, such as post-spawning periods [,,]. This mechanism could or could not have an effect on the distribution of DA in the different areas of the digestive gland. The third possibility is that the intestinal loop cells which contain the toxin derive from cells from the digestive gland. If the new cells of the intestinal loop are generated in the nearby area of the digestive gland, a reduction in the toxin concentration in that area would be expected. Intestinal cells are mostly epithelial and the presence of immature cells, as in the digestive gland, has not been observed []. It is therefore possible that the intestinal cells derive from immature cells in the digestive gland and consequently inherit the toxin they contain. This would explain the lower DA concentration in the sector closest to the gonad and also the greater reduction in the outer region relative to the inner one, because the cells in this region are the closest to the intestinal loop. The confirmation of this hypothesis requires additional studies.

3. Discussion

Domoic acid was preferentially retained in large cells, mostly absorptive (digestive cells) []. The concentration measured in small cells, mostly secretory and immature cells, was substantially lower than in the digestive ones. In terms of toxin burden, the importance of large cells was even greater, because they are found in a larger amount in the digestive gland (more biomass). The difference in concentration opens two possibilities regarding the methods of depuration not taken into account to date: (a) the secretory cells depurate the DA they have accumulated during the intoxication phase (which could be the case if both cell types accumulate the toxin to the same extent); and (b) DA is transferred from absorptive to secretory cells, which excrete it. Unfortunately, no information about the distribution of DA during the intoxication phase exists and the transfer between cellular types has not been documented, so new studies would be required to shed some light on the subject. However, in our opinion, neither of the two ways is very likely. Firstly, it seems unlikely that absorptive and secretory cells would absorb and accumulate DA during the intoxication at the same rate, because of their main functions. Secondly, it is difficult to explain how the toxin could pass the cellular membrane of the absorptive cells to go to the secretory ones but not to be excreted (the depuration rate is very low [,]) or transferred to other tissues [,].