Inhibitory effects of chlorophyll pigments on the bioaccessibility of ß-carotene: Influence of chlorophyll structure and oil matrix.

Chlorophylls and ß-carotene are fat-soluble phytochemicals in daily diets, while their bioaccessibility interaction remains unknown. Eight dietary chlorophylls and their derivatives (chlorophyll a, chlorophyll b, pheophytin a, pheophytin b, chlorophyllide a, chlorophyllide b, pheophorbide a, pheophorbide b) were combined with ß-carotene in six different oil matrices (corn oil, coconut oil, medium-chain triglycerides, peanut oil, olive oil and fish oil) and were subjected to in vitro digestion. Generally, chlorophylls significantly decreased ß-carotene bioaccessibility by competitive incorporation into micelles. Dephytylated chlorophylls had a greater inhibitory effect on the micellarization and bioaccessibility of ß-carotene compared to phytylated chlorophylls. In their co-digestion system, olive oil group exhibited the smallest particle size and biggest zeta potential in both digesta and micelles. For chlorophylls, the phytol group and their levels are key factors, which was also buttressed by the mice model where additional supplementation of pheophorbide a significantly hindered the accumulation of ß-carotene and retinoids compounds.

Elevated temperature extraction of ß-carotene from freeze-dried carrot powder into sunflower oil: Extraction kinetics and thermal stability.

ß-Carotene, a precursor of vitamin A, can alleviate the deficiency of this vitamin prevalent worldwide. Earlier research studies have addressed the extraction of ß-carotene at relatively low temperatures (up to 70°C) due to its perceived instability at higher temperatures, as a result of which extraction rates recorded are relatively low. This study models the net rate of ß-carotene extraction by considering both extraction and degradation kinetics. The model developed, which accounts for degradation occurring in solid and extract phases, has been experimentally validated for the extraction of ß-carotene from freeze-dried carrot powder into sunflower oil over a range of temperatures 90-150°C. This study also gives insights into the application of sunflower oil as a carrier for ß-carotene during cooking and food processing, by monitoring and modeling the thermal degradation and isomerization of ß-carotene at temperatures up to 220°C. The modeling of extraction kinetics shows that it is possible to achieve viable extraction rates by employing temperatures in the range (90-150°C) for relatively short times (<5 min). The degradation kinetics shows that almost 75% of the ß-carotene can survive heating at 180°C for 10 min-indicating the possibility of using ß-carotene enriched edible oils for frying. This study also reports on the formation of three isomers of ß-carotene identified using HPLC: trans-, 9-cis, and 13-cis. The reaction network model developed in this study was able to account for the transient variation of the concentration of all three isomers. PRACTICAL APPLICATION: ß-Carotene is a precursor of vitamin A and its consumption can potentially alleviate the deficiency of this vitamin prevalent worldwide. This study validates a model for the extraction of ß-carotene in sunflower oil, which takes into account extraction as well as degradation occurring during extraction, so that a rational method is available for the design of efficient extractors for this purpose. This paper also establishes the thermal stability of ß-carotene under frying conditions by quantifying its thermal degradation as well as isomerization.

Analysis of the relationship between color and natural pigments of tobacco leaves during curing.

Color is one of the most important indicators for the flue-cured tobacco quality. The color change of tobacco has a great relationship with the natural pigments in the tobacco. The relationship between color characteristics and the content of natural pigments in tobacco leaves during curing was investigated. The middle part of variety K326 tobacco was taken at each key time point during the curing process to determine the changes of color characteristics, moisture, pigment and polyphenol content. The results showed that moisture content of wet basis of tobacco gradually decreased from 72 to 18% during the curing process, the b* value increased and then decreased, and the a* value increased significantly. The lutein and ß-carotene content decreased to 63.83 µg/g and 28.3 µg/g, respectively. The total polyphenols content increased to 50.19 mg/g. Meanwhile, the a* value was significantly and positively correlated with polyphenols content and negatively correlated with pigments content. Cluster analysis showed that the samples were divided into three categories: samples with the curing time of 0 h, 24-72 h, and 84-132 h. These results demonstrated that the color change of tobacco during curing process can be divided into three stages from the perspective of chemical composition, which are strongly related to the degradation of pigments and the transformation of polyphenols.

ß-carotene inhibits MAPKs signaling pathways on rat colonic epithelial cells to attenuate TNF-α-induced intestinal inflammation and injury.

This experiment successfully isolated the rat colonic epithelial cells and established a TNF-α-induced intestinal inflammation model. Western Blot was used to detect the related protein expression levels of the MAPKs signaling pathway. QPCR technology was used to detect the expression of aquaporins, intestinal mucosal repair factor, and inflammatory factors. The results show that 25 µM ß-carotene pretreatment at 24 h can inhibit MAPKs signaling pathway activated by TNF-α, change the relative mRNA expression of inflammatory cytokines, intestinal mucosal repair factors, and aquaporins, and the phosphorylated protein expression of p38, ERK, and NF-κB were attenuated to reduce inflammatory damage. After inhibiting p38 and ERK, the effect of ß-carotene was reduced significantly (P < 0.05). In conclusion, ß-carotene can alleviate the abnormal expression of aquaporins caused by inflammation through the MAPKs signaling pathway. This is for ß-carotene as a functional nutrient that provides new insights.

Circulating microRNAs as novel biomarkers for measuring the potency of ginger extract against cyclophosphamide toxicity in rat renal tissues: molecular and histopathological study.

OBJECTIVE: This study aims to explore underlying molecular variations in the expression of miRNAs in kidney tissues of ginger-treated and non-treated cyclophosphamide (CP)-intoxicated rats. MATERIALS AND METHODS:   A total of 40 adult male Wistar rats were randomly divided into four groups of 10 each: Group I (control: received normal food and water), Group II (received ginger at a dose of 300 mg/kg), Group III (received CP 75 mg/kg, i.p.), and Group IV (received the same dose of CP and ginger extract).  Rats received a single injection of 75 mg/kg CP on days 3, 4, 5, 19, 20, and 21. In CP-intoxicated rats, the treatment with ginger extract at a dose of 300 mg/kg was received by oral gavage starting seven days before CP and continuing throughout the duration of the experiment for four weeks. Molecular variations in the expression of miRNAs, apoptotic genes, histological kidney damage, and abnormal kidney function in control, ginger, and CP-intoxicated rats were identified by using real-time RT-PCR Analysis, immunohistochemical, and colorimetric assays. In addition, HPLC analysis and liquid chromatography spectrophotometry analysis using Diphenyl-1-picrylhydrazyl (DPPH) radical, and Β-Carotene-linoleic acid reagents were applied respectively for in-vitro screening of phytoconstituents and antioxidant activity for ginger extract. RESULTS: The kidney tissues of CP-intoxicated rats displayed an increase in lipid peroxidation marker malonaldehyde (MDA), DNA damage, and fibrosis markers like hyaluronic acid (HA) and hydroxyproline Hypx) with a decrease in the superoxide dismutase (SOD) and total antioxidant capacity (TAC). In addition, molecular expressions of mRNA fibrotic genes such as collagen, type 1, alpha 1 (COL1A1), and α-smooth muscle actin (αSMA). Molecular expressions of levels of B-cell lymphoma 2 (BCl-2) mRNA gene were down-regulated, and the expression of mRNA apoptotic; BCL2 associated X gene (Bax), caspase-3, Bax/BCl-2 ratio genes were significantly up-regulated respectively. Moreover, cellular oxidative genes, erythroid 2-related factor (Nrf2), and heme oxygenase-1 (HO-1) were down-regulated, respectively. The miR-155-5p, miR-34a-5p, miR-21-5p significantly increased while the miR-193b-3p, miR-455-3p, and miR-342-3p significantly decreased. Ginger also increased the expression of Nrf2, HO-1, and BCl-2 genes in the kidneys of rats induced with CP. In addition, active phytoconstituents, particularly 6]]-shogaol and 6]]-gingerol, were significantly identified in ginger extract using HPLC analysis. Antioxidant activity of these active metabolites were shown to be higher against in vitro free radicals (DPPH and Β-Carotene-linoleic acid), suggesting the potential antioxidant and antiapoptotic properties of ginger against CP-toxicity. CONCLUSIONS: Treatment with ginger in rats induced with CP resulted in significant improvement in the expression of certain molecular miRNAs. The kidney tissues of these rats showed a marked decrease in the expression of miR-155-5p, miR-34a-5p, and miR-21-5p, while the levels of miR-193b-3p, miR-455-3p, and miR-342-3p were observed to increase significantly. In conclusion, ginger can protect rats from CP-induced nephrotoxicity.

Retinoic Acid-PPARα Mediates ß-Carotene Resistance to Placental Dysfunction Induced by Deoxynivalenol.

Deoxynivalenol (DON), one of the most polluted mycotoxins in the environment and food, has been proven to have strong embryonic and reproductive toxicities. However, the effects of DON on placental impairment and effective interventions are still unclear. This study investigated the effect of ß-carotene on placental functional impairment and its underlying molecular mechanism under DON exposure. Adverse pregnancy outcomes were caused by intraperitoneal injection of DON from 13.5 to 15.5 days of gestation in mice, resulting in higher enrichment of DON in placenta than in other tissue samples. Interestingly, 0.1% ß-carotene dietary supplementation could significantly alleviate DON-induced pregnancy outcomes. Additionally, in vivo and in vitro placental barrier models demonstrated the association of DON-induced placental function impairment with placental permeability barrier disruption, angiogenesis impairment, and oxidative stress induction. Moreover, ß-carotene regulated DON-induced placental toxicity by activating the expressions of claudin 1, zonula occludens-1, and vascular endothelial growth factor-A through retinoic acid-peroxisome proliferator-activated receptor α signaling.

Effects of long-term blue light irradiation on carotenoid biosynthesis and antioxidant activities in Chinese cabbage (Brassica rapa L. ssp. pekinensis).

The aim of this study was to investigate the impact of long-term exposure to blue light-emitting diodes (LEDs) on the accumulation of indolic glucosinolates and carotenoids, as well as the plant growth and antioxidant activities in both orange and common Chinese cabbage (Brassica rapa L. ssp. pekinensis). Blue light treatment also induced higher ferric-reducing antioxidant power and 2,2-diphenyl-1-picrylhydrazyl by 20.66 % and 30.82 % and antioxidant enzyme activities catalase, peroxidase, superoxide dismutase, and the accumulation of non-enzymatic antioxidant substances (total phenols and total flavonoids) in the orange Chinese cabbage. Furthermore, long-term exposure to blue light had negative effects on the net photosynthetic rate and chlorophyll fluorescence levels. Meanwhile, blue light promoted accumulation of Indol-3-ylmethyl glucosinolate (I3M), ß-carotene, lutein and zeaxanthin due to the high expression of regulatory and biosynthetic genes of the above metabolic pathways. In particular, lycopene and ß-carotene content in orange Chinese cabbage increased by 60.14 % and 65.33 % compared to the ones in common line. The accumulation of carotenoid and increasing antioxidant levels in the orange cabbage line was influenced by long-term blue light irradiation, leading to better tolerance to low temperature and drought stresses. The up-regulation of transcription factors such as BrHY5-2, BrPIF4 and BrMYB12 may also contribute to the increased tolerance in orange Chinese cabbage to extreme environmental stresses. The BrHY5-2 gene could activate carotenoid biosynthetic genes and induce the accumulation of carotenoids. These findings suggested that long-term blue light irradiation could be a promising technique for increasing the nutrition value and enhancing tolerance to low temperature and drought stresses in Chinese cabbage.

Functional Characterization of Lycopene ß- and ε-Cyclases from a Lutein-Enriched Green Microalga Chlorella sorokiniana FZU60.

Lutein is a high-value carotenoid with many human health benefits. Lycopene ß- and ε-cyclases (LCYB and LCYE, respectively) catalyze the cyclization of lycopene into distinct downstream branches, one of which is the lutein biosynthesis pathway, via α-carotene. Hence, LCYB and LCYE are key enzymes in lutein biosynthesis. In this study, the coding genes of two lycopene cyclases (CsLCYB and CsLCYE) of a lutein-enriched marine green microalga, Chlorella sorokiniana FZU60, were isolated and identified. A sequence analysis and computational modeling of CsLCYB and CsLCYE were performed using bioinformatics to identify the key structural domains. Further, a phylogenetic analysis revealed that CsLCYB and CsLCYE were homogeneous to the proteins of other green microalgae. Subcellular localization tests in Nicotiana benthamiana showed that CsLCYB and CsLCYE localized in chloroplasts. A pigment complementation assay in Escherichia coli revealed that CsLCYB could efficiently ß-cyclize both ends of lycopene to produce ß-carotene. On the other hand, CsLCYE possessed a strong ε-monocyclase activity for the production of δ-carotene and a weak ε-bicyclic activity for the production of ε-carotene. In addition, CsLCYE was able to catalyze lycopene into ß-monocyclic γ-carotene and ultimately produced α-carotene with a ß-ring and an ε-ring via γ-carotene or δ-carotene. Moreover, the co-expression of CsLCYB and CsLCYE in E. coli revealed that α-carotene was a major product, which might lead to the production of a high level of lutein in C. sorokiniana FZU60. The findings provide a theoretical foundation for performing metabolic engineering to improve lutein biosynthesis and accumulation in C. sorokiniana FZU60.

Overexpressing Carotenoid Biosynthetic Genes in Synechocystis sp. PCC 6803 Improved Intracellular Pigments and Antioxidant Activity, Which Can Decrease the Viability and Proliferation of Lung Cancer Cells In Vitro.

In the antioxidant system in cyanobacteria, non-enzymatic antioxidants, such as carotenoids, are considered good candidates for coping with oxidative stress, particularly light stress, and pharmaceutical therapeutic applications. A significant amount of carotenoid accumulation has been recently improved by genetic engineering. In this study, to achieve higher carotenoid production with higher antioxidant activity, we successfully constructed five Synechocystis sp. PCC 6803 strains overexpressing (OX) native genes related to the carotenoids biosynthetic pathway, including OX_CrtB, OX_CrtP, OX_CrtQ, OX_CrtO, and OX_CrtR. All of the engineered strains maintained a significant quantity of myxoxanthophyll, while increasing zeaxanthin and echinenone accumulation. In addition, higher components of zeaxanthin and echinenone were noted in all OX strains, ranging from 14 to 19% and from 17 to 22%, respectively. It is worth noting that the enhanced echinenone component responded to low light conditions, while the increased ß-carotene component contributed to a high light stress response. According to the higher antioxidant activity of all OX strains, the carotenoid extracts presented lower IC50 in lung cancer cell lines H460 and A549, with values less than 157 and 139 µg/mL, respectively, when compared with those of WTc, particularly OX_CrtR and OX_CrtQ. A higher proportion of zeaxanthin and ß-carotene in OX_CrtR and OX_CrtQ, respectively, may considerably contribute to the ability to treat lung cancer cells with antiproliferative and cytotoxic effects.

Emulsifying properties, in vitro digestive characteristics, and ß-carotene bioaccessibility of mandarin peel pectin emulsions prepared with different carrier oil phases.

Mandarin peel pectin (MPP) emulsions were prepared with different oil phase loadings with or without ß-carotene, and their emulsifying characteristics, digestive properties and ß-carotene bioaccessibility were investigated. Results revealed that all MPP emulsions exhibited good loading efficiency for ß-carotene, while their apparent viscosity and interfacial pressure (π) of MPP emulsions increased significantly after the addition of ß-carotene. Emulsification of MPP emulsions as well as digestibility were significantly dependent on the kind of oil. MPP emulsions prepared with long-chain triglycerides (LCT) oil (soybean, corn, and olive oil) exhibited higher volume average particle size (D4,3), apparent viscosity, π values, and bioaccessibility of carotene compared to those prepared with medium-chain oils (MCT). MPP emulsions with LCT rich in monosaturated fatty acids (olive oil) had the highest ß-carotene encapsulation efficiency, bioaccessibility, etc. than from other oils. This study provides a theoretical basis for the efficient encapsulation and high bioaccessibility of carotenoids with pectin emulsions.

ß-Carotene induces UCP1-independent thermogenesis via ATP-consuming futile cycles in 3T3-L1 white adipocytes.

The activation of brown fat and induction of beige adipocytes, so-called non-shivering thermogenesis, is emerging as a promising target for therapeutic intervention in obesity management. Our previous report demonstrated that ß-carotene (BC) induces beige adipocytes to increase UCP1-dependent thermogenic activity. However, the UCP1-independent thermogenic effect of BC on adipose tissues remains unexplored. In this study, we examined the effects of BC on UCP1-independent thermogenic activity with a focus on the ATP-consuming futile cycles in 3T3-L1 adipocytes. BC increased intracellular calcium levels and stimulated the expression of calcium cycling-related proteins, including sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) 2b, ryanodine receptor 2 (RyR2), voltage-dependent anion channel (VDAC), mitochondrial calcium uniporter (MCU), and Ca2+/calmodulin-dependent protein kinase 2 (CaMK2) in 3T3-L1 white adipocytes. In addition, BC stimulated thermogenesis by activating the creatine metabolism-related thermogenic pathway. Moreover, BC activated ß-carotene oxygenase 1 (BCO1), which efficiently cleaved BC to retinal and consequently converted to its transcriptionally active form retinoic acid. These BC conversion products also exhibited thermogenic effects comparable to a similar level of BC. The mechanistic study revealed that retinal exhibited thermogenic activity independently of retinoic acid and retinoic acid-mediated thermogenesis was resulted partly from conversion of retinal. Moreover, BC activated α1-AR and UCP1-independent thermogenic effectors independently of UCP1 expression. In conclusion, the thermogenic response to BC and its conversion products in 3T3-L1 white adipocytes involves two interacting pathways, one mediated via ß3-adrenergic receptors (ß3-AR) and cyclic adenosine monophosphate (cAMP) and the other via α1-AR and increases in cytosolic Ca2+ levels activated by calcium regulatory proteins.

Combining ß-Carotene with 5-FU via Polymeric Nanoparticles as a Novel Therapeutic Strategy to Overcome uL3-Mediated Chemoresistance in p53-Deleted Colorectal Cancer Cells.

Colorectal cancer (CRC) is one of the leading causes of cancer-related death worldwide. Despite recent therapeutic advancements, resistance to 5-fluorouracil (5-FU) remains a major obstacle to the successful treatment of this disease. We have previously identified the ribosomal protein uL3 as a key player in the cell response to 5-FU, and loss of uL3 is associated with 5-FU chemoresistance. Natural products, like carotenoids, have shown the ability to enhance cancer cell response to drugs and may provide a safer choice to defeat chemoresistance in cancer. Transcriptome analysis of a cohort of 594 colorectal patients revealed a correlation between uL3 expression and both progression-free survival and response to treatment. RNA-Seq data from uL3-silenced CRC cells demonstrated that a low uL3 transcriptional state was associated with an increased expression of specific ATP-binding cassette (ABC) genes. Using two-dimensional (2D) and three-dimensional (3D) models of 5-FU-resistant CRC cells stably silenced for uL3, we investigated the effect of a novel therapeutic strategy by combining ß-carotene and 5-FU using nanoparticles (NPs) as a drug delivery system. Our results indicated that the combined treatment might overcome 5-FU chemoresistance, inducing cell cycle arrest in the G2/M phase and apoptosis. Furthermore, the combined treatment significantly reduced the expression levels of analyzed ABC genes. In conclusion, our findings suggest that ß-carotene combined with 5-FU may be a more effective therapeutic approach for treating CRC cells with low levels of uL3.

ETHYLENE-INSENSITIVE 3-LIKE 2 regulates ß-carotene and ascorbic acid accumulation in tomatoes during ripening.

ETHYLENE-INSENSITIVE 3/ETHYLENE-INSENSITIVE 3-LIKEs (EIN3/EILs) are important ethylene response factors during fruit ripening. Here, we discovered that EIL2 controls carotenoid metabolism and ascorbic acid (AsA) biosynthesis in tomato (Solanum lycopersicum). In contrast to the red fruits presented in the wild type (WT) 45 d after pollination, the fruits of CRISPR/Cas9 eil2 mutants and SlEIL2 RNA interference lines (ERIs) showed yellow or orange fruits. Correlation analysis of transcriptome and metabolome data for the ERI and WT ripe fruits revealed that SlEIL2 is involved in ß-carotene and AsA accumulation. ETHYLENE RESPONSE FACTORs (ERFs) are the typical components downstream of EIN3 in the ethylene response pathway. Through a comprehensive screening of ERF family members, we determined that SlEIL2 directly regulates the expression of 4 SlERFs. Two of these, SlERF.H30 and SlERF.G6, encode proteins that participate in the regulation of LYCOPENE-ß-CYCLASE 2 (SlLCYB2), encoding an enzyme that mediates the conversion of lycopene to carotene in fruits. In addition, SlEIL2 transcriptionally repressed L-GALACTOSE 1-PHOSPHATE PHOSPHATASE 3 (SlGPP3) and MYO-INOSITOL OXYGENASE 1 (SlMIOX1) expression, which resulted in a 1.62-fold increase of AsA via both the L-galactose and myoinositol pathways. Overall, we demonstrated that SlEIL2 functions in controlling ß-carotene and AsA levels, providing a potential strategy for genetic engineering to improve the nutritional value and quality of tomato fruit.

ß-Cryptoxanthin Attenuates Cigarette-Smoke-Induced Lung Lesions in the Absence of Carotenoid Cleavage Enzymes (BCO1/BCO2) in Mice.

High dietary intake of ß-cryptoxanthin (BCX, an oxygenated provitamin A carotenoid) is associated with a lower risk of lung disease in smokers. BCX can be cleaved by ß-carotene-15,15'-oxygenase (BCO1) and ß-carotene-9',10'-oxygenase (BCO2) to produce retinol and apo-10'-carotenoids. We investigated whether BCX has protective effects against cigarette smoke (CS)-induced lung injury, dependent or independent of BCO1/BCO2 and their metabolites. Both BCO1-/-/BCO2-/- double knockout mice (DKO) and wild type (WT) littermates were supplemented with BCX 14 days and then exposed to CS for an additional 14 days. CS exposure significantly induced macrophage and neutrophil infiltration in the lung tissues of mice, regardless of genotypes, compared to the non-exposed littermates. BCX treatment significantly inhibited CS-induced inflammatory cell infiltration, hyperplasia in the bronchial epithelium, and enlarged alveolar airspaces in both WT and DKO mice, regardless of sex. The protective effects of BCX were associated with lower expression of IL-6, TNF-α, and matrix metalloproteinases-2 and -9. BCX treatment led to a significant increase in hepatic BCX levels in DKO mice, but not in WT mice, which had significant increase in hepatic retinol concentration. No apo-10'-carotenoids were detected in any of the groups. In vitro BCX, at comparable doses of 3-OH-ß-apo-10'-carotenal, was effective at inhibiting the lipopolysaccharide-induced inflammatory response in a human bronchial epithelial cell line. These data indicate that BCX can serve as an effective protective agent against CS-induced lung lesions in the absence of carotenoid cleavage enzymes.

ß-Carotene suppresses cancer cachexia by regulating the adipose tissue metabolism and gut microbiota dysregulation.

Cancer cachexia is a metabolic disease affecting multiple organs and characterized by loss adipose and muscle tissues. Metabolic dysregulated of adipose tissue has a crucial role in cancer cachexia. ß-Carotene (BC) is stored in adipose tissues and increases muscle mass and differentiation. However, its regulatory effects on adipose tissues in cancer cachexia have not been investigated yet. In this study, we found that BC supplementations could inhibit several cancer cachexia-related changes, including decreased carcass-tumor (carcass weight after tumor removal), adipose weights, and muscle weights in CT26-induced cancer cachexia mice. Moreover, BC supplementations suppressed cancer cachexia-induced lipolysis, fat browning, hepatic gluconeogenesis, and systemic inflammation. Altered diversity and composition of gut microbiota in cancer cachexia were restored by the BC supplementations. BC treatments could reverse the down-regulated adipogenesis and dysregulated mitochondrial respiration and glycolysis in adipocytes and colon cancer organoid co-culture systems. Taken together, these results suggest that BC can be a potential therapeutic strategy for cancer cachexia.

The conversion of ß-carotene to vitamin A in adipocytes drives the anti-obesogenic effects of ß-carotene in mice.

OBJECTIVE: The ß-carotene oxygenase 1 (BCO1) is the enzyme responsible for the cleavage of ß-carotene to retinal, the first intermediate in vitamin A formation. Preclinical studies suggest that BCO1 expression is required for dietary ß-carotene to affect lipid metabolism. The goal of this study was to generate a gene therapy strategy that over-expresses BCO1 in the adipose tissue and utilizes the ß-carotene stored in adipocytes to produce vitamin A and reduce obesity. METHODS: We generated a novel adipose-tissue-specific, adeno-associated vector to over-express BCO1 (AT-AAV-BCO1) in murine adipocytes. We tested this vector using a unique model to achieve ß-carotene accumulation in the adipose tissue, in which Bco1-/- mice were fed ß-carotene. An AT-AAV over-expressing green fluorescent protein was utilized as control. We evaluated the adequate delivery route and optimized cellular and organ specificity, dosage, and exposure of our vectors. We also employed morphometric analyses to evaluate the effect of BCO1 expression in adiposity, as well as HPLC and mass spectrometry to quantify ß-carotene and retinoids in tissues, including retinoic acid. RESULTS: AT-AAV-BCO1 infusions in the adipose tissue of the mice resulted in the production of retinoic acid, a vitamin A metabolite with strong effects on gene regulation. AT-AAV-BCO1 treatment also reduced adipose tissue size and adipocyte area by 35% and 30%, respectively. These effects were sex-specific, highlighting the complexity of vitamin A metabolism in mammals. CONCLUSIONS: The over-expression of BCO1 through delivery of an AT-AAV-BCO1 leads to the conversion of ß-carotene to vitamin A in adipocytes, which subsequently results in reduction of adiposity. These studies highlight for the first time the potential of adipose tissue ß-carotene as a target for BCO1 over-expression in the reduction of obesity.

Characteristics of a new carotenoid cleavage dioxygenase NtCCD10 derived from Nicotiana tabacum.

MAIN CONCLUSION: A new carotenoid cleavage dioxygenase NtCCD10 from tobacco was characterized. There is some difference between NtCCD10 and CCD1 in structure. NtCCD10 can cleave the C5-C6 (C5'-C6') and C9-C10 (C9'-C10') double bonds of carotenoids and has high catalytic activity. Carotenoid cleavage dioxygenases (CCDs) cleave carotenoids to produce a variety of apocarotenoids, which have important biological functions for organisms in nature. There are eleven CCDs subfamilies in the plant kingdom, many of which have been extensively characterized in their functions. However, as a newly classified subfamily, the function of CCD10 has rarely been studied. In this work, the function of an NtCCD10 gene from dicotyledonous Nicotiana tabacum was cloned and characterized, and its phylogeny, molecular structural modeling and protein structure were also systematically analyzed. Like other CCDs, NtCCD10 also possesses a seven bladed ß-propeller with Fe2+ cofactor in its center constituting the active site of the enzyme. The Fe2+ is also coordinated bonding with four conserved histidine residues. Meanwhile, NtCCD10 also has many unique features, such as its α1 and α3 helixes are not anti-parallel, a special ß-sheet and a longer access tunnel for substrates. When expressed in engineered Escherichia coli (producing phytoene, lycopene, ß-carotene, and zeaxanthin) and Saccharomyces cerevisiae (producing ß-carotene), NtCCD10 could symmetrically cleave phytoene and ß-carotene at the C9-C10 and C9'-C10' positions to produce geranylacetone and ß-ionone, respectively. In addition, NtCCD10 could also cleave the C5-C6 and C5'-C6' double bonds of lycopene to generate 6-methyl-5-heptene-2-one (MHO). NtCCD10 has higher catalytic activity than PhCCD1 in yeast, which provides a good candidate CCD for biosynthesis of ß-ionone and has potential applications in biotechnological industry. This study identified the taxonomic position and catalytic activity of the first NtCCD10 in dicotyledonous plants. This will provide a reference for the discovery and functional identification of CCD10 enzymes in dicotyledons.

Torularhodin Alleviates Hepatic Dyslipidemia and Inflammations in High-Fat Diet-Induced Obese Mice via PPARα Signaling Pathway.

Torularhodin is a ß-carotene-like compound from Sporidiobolus pararoseus, and its protective effect against high-fat diet (HFD)-induced hepatic dyslipidemia and inflammation was investigated. Compared to mice of C57BL/6J fed on HFD, the addition of Torularhodin into the HFD (HFD-T) significantly reduced body weight, serum triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL), and the inflammatory mediators of TNF-α, IL-6, IL-1ß, and lipopolysaccharide (LPS). A significant increase of high-density lipoprotein cholesterol (HDL-c), which is beneficial to cholesterol clearance, was also observed in HFD-T group. Proteomic analysis showed HDL-C-c is highly correlated with proteins (e.g., CPT1A and CYP7A1) involved in lipid ß-oxidation and bile acid synthesis, whereas the other phenotypic parameters (TC, TG, LDL, and inflammatory cytokines) are highly associated with proteins (e.g., SLC27A4) involved in lipid-uptake. The up-regulated anti-inflammation proteins FAS, BAX, ICAM1, OCLN, GSTP1, FAF1, LRP1, APEX1, ROCK1, MANF, STAT3, and INSR and down-regulated pro-inflammatory proteins OPTN, PTK2B, FADD, MIF, CASP3, YAP1, DNM1L, and NAMPT not only demonstrate the occurrence of HFD-induced hepatic inflammation, but also prove the anti-inflammatory property of Torularhodin. KEGG signaling pathway analysis revealed that the PPARα signaling pathway is likely fundamental to the health function of Torularhodin through up-regulating genes related to fatty acid ß-oxidation, cholesterol excretion, HDL-Cc formation, and anti-inflammation. Torularhodin, as a new food resource, may act as a therapeutic agent to prevent hepatic dyslipidemia and related inflammation for improved health.

Advances in the synthesis of three typical tetraterpenoids including ß-carotene, lycopene and astaxanthin.

Carotenoids are natural pigments that widely exist in nature. Due to their excellent antioxidant, anticancer and anti-inflammatory properties, carotenoids are commonly used in food, medicine, cosmetic and other fields. At present, natural carotenoids are mainly extracted from plants, algae and microorganisms. With the rapid development of metabolic engineering and molecular biology as well as the continuous in-depth study of carotenoids synthesis pathways, industrial microorganisms have showed promising applications in the synthesis of carotenoids. In this review, we introduced the properties of several carotenoids and their biosynthetic metabolism process. Then, the microorganisms synthesizing carotenoids through the natural and non-natural pathways and the extraction methods of carotenoids were summarized and compared. Meanwhile, the influence of substrates on the carotenoids production was also listed. The methods and strategies for achieving high carotenoid production are categorized to help with future research.

ß-Carotene from the Alga Dunaliella bardawil Decreases Gene Expression of Adipose Tissue Macrophage Recruitment Markers and Plasma Lipid Concentrations in Mice Fed a High-Fat Diet.

Vitamin A and provitamin A carotenoids are involved in the regulation of adipose tissue metabolism and inflammation. We examined the effect of dietary supplementation using all-trans and 9-cis ß-carotene-rich Dunaliella bardawil alga as the sole source of vitamin A on obesity-associated comorbidities and adipose tissue dysfunction in a diet-induced obesity mouse model. Three-week-old male mice (C57BL/6) were randomly allocated into two groups and fed a high-fat, vitamin A-deficient diet supplemented with either vitamin A (HFD) or ß-carotene (BC) (HFD-BC). Vitamin A levels in the liver, WATs, and BAT of the HFD-BC group were 1.5-2.4-fold higher than of the HFD group. BC concentrations were 5-6-fold greater in BAT compared to WAT in the HFD-BC group. The eWAT mRNA levels of the Mcp-1 and Cd68 were 1.6- and 2.1-fold lower, respectively, and the plasma cholesterol and triglyceride concentrations were 30% and 28% lower in the HFD-BC group compared with the HFD group. Dietary BC can be the exclusive vitamin A source in mice fed a high-fat diet, as shown by the vitamin A concentration in the plasma and tissues. Feeding BC rather than vitamin A reduces adipose tissue macrophage recruitment markers and plasma lipid concentrations.