BC Supplementation Research Articles

Effect of β-caryophyllene on Sperm Cryopreservation.

This study aimed to assess the impact of β-caryophyllene (BC) supplementation in the extender on the post-cryopreservation quality of semen from Dorper rams. Six Dorper rams were utilized for semen collection over 16 weeks, with BC concentrations determined via the MTT test. Animals were divided into a control group and three treatment groups receiving BC at concentrations of 1.0mM, 2.0mM, and 3.0mM in the Trisegg yolk diluent. Semen was cryopreserved and stored in liquid nitrogen for at least 15 days. After thawing, in vitro assessments including CASA, acrosomal integrity, plasma membrane integrity, mitochondrial membrane potential, and thermo-resistance tests were conducted. Additionally, the TBARS assay was performed to evaluate oxidative stress. While BC supplementation did not significantly affect sperm motility, it notably improved mitochondrial potential and mitigated oxidative stress in cryopreserved ram semen. Incorporating β-caryophyllene into the extender exhibited beneficial effects on the quality of Dorper ram semen post-cryopreservation, enhancing mitochondrial functionality and reducing oxidative stress.

β‑carotene attenuates muscle wasting in cancer cachexia by regulating myogenesis and muscle atrophy.

Cancer cachexia is a metabolic disease involving multiple organs, which is accompanied by the depletion of muscle tissue and is associated with ~20% of cancer‑related deaths. Muscle wasting is a critical factor in cancer cachexia. β‑carotene (BC) has been shown to increase muscle mass and hypertrophy in healthy mice. However, its effects on muscle tissue dysregulation in cancer cachexia have yet to be studied. In the present study, 5‑week‑old male C57BL/6J mice were injected with 1x106 Lewis lung carcinoma (LLC) cells to induce cancer cachexia; then the mice were administered BC (4or8mg/kg) for 22days to assess its effects on muscle atrophy in the gastrocnemius muscles. The effects of BC on inflammatory cytokines, myogenesis and muscle atrophy were evaluated using C2C12 myotubes treated with LLC‑conditioned media. BC supplementation significantly suppressed tumor growth, inflammatory cytokines, and hepatic gluconeogenesis in the LLC‑induced cancer cachexia mouse model, while also improving muscle weight and grip strength. These effects are considered to be mediated by the PI3K/Akt pathway and through regulation of muscle atrophy. Moreover, BC treatment was associated with the recovery of LLC‑conditioned media‑induced muscle differentiation deficits and muscle atrophy in C2C12 myotubes. These findings indicate BC as a potential novel therapeutic agent for cancer cachexia.

The clinical significance of low dose biotin supplements (

BackgroundIn the last decade, the combination of the widespread use of streptavidin–biotin technology and biotin–containing supplements (BCS) in the daily clinical practice, have led to numerous reports of erroneous hormone immunoassay results. However, there are no studies assessing the clinical and biochemical significance of that phenomenon, when treating patients with hypothyroidism. Therefore, a prospective study was designed to investigate the potential alterations in the measurement of thyroid hormone concentrations and clinical consequences in patients with hypothyroidism using low -dose BCS containing less than 300 μg/day.MethodsFifty-seven patients on thyroxine supplementation, as a result of hypothyroidism and concurrent use of BCS at a dose <300μg/day for 10 to 60 days were prospectively evaluated. Namely, TSH and free T4 (FT4) concentration measurements were performed, during BC supplementation and 10 days post BCS discontinuation and compared to 31 age-matched patients with supplemented hypothyroidism and without BCS.ResultsA statistically significant increase in TSH and decline in FT4 concentrations was observed after BCS discontinuation. However, on clinical grounds, these modifications were minor and led to medication dose adjustment in only 2/57 patients (3.51%) in whom TSH was notably decreased after supplement discontinuation.ConclusionOur study suggests that changes in thyroid hormones profiling, due to supplements containing low dose biotin, are of minimal clinical relevance and in most cases don’t occult the need to adjust the thyroxine replacement dose in patients with hypothyroidism. Larger, well-designed trials are required to further evaluate this phenomenon.

Alkalihalobacillus clausii (formerly Bacillus clausii) spores lessen antibiotic-induced intestinal injury and reshape gut microbiota composition in mice

The antibiotic-induced intestinal injury (AIJ) is associated with diarrhoea and gastrointestinal discomfort. However, the pathological intestinal mechanisms and related side effects associated with antibiotic use/misuse may be counteracted by probiotics. This study aims to evaluate the effect and the protective mechanisms of a probiotic formulation containing Alkalihalobacillus clausii (formerly Bacillus clausii; BC) spores in an experimental model of AIJ. C57/Bl6J mice were orally challenged with a high dose of ceftriaxone for five days along with BC treatment which lasted up to the 15th day. Our results showed the beneficial effect of the probiotic in preserving colonic integrity and limiting tissue inflammation and immune cell infiltration in AIJ mice. BC increased tight junction expression and regulated the unbalanced production of colonic pro- and anti-inflammatory cytokines, converging toward the full resolution of the intestinal damage. These findings were supported by the histological evaluation of the intestinal mucosa, suggesting a potential restoration of mucus production. Notably, BC treatment increased gene transcription of the secretory products responsible for epithelium repair and mucus synthesis and normalized the expression of antimicrobial peptides involved in immune activation. Reconstruction of complex and diverse gut microbiota in antibiotic-induced dysbiosis was recorded upon BC supplementation. Specifically, the expansion of A. clausii, Prevotella rara and Eubacterium ruminatium drove intestinal microbiota rebalance by primarily impacting Bacteroidota members. Taken together, our data indicate that BC administration alleviates AIJ by multiple converging mechanisms leading to restoring gut integrity and homeostasis and reshaping microbiota composition.

Bovine Colostrum Supplementation Modulates the Intestinal Microbial Community in Rabbits.

BC is a nutraceutical that can modulate intestinal microbiota. This study investigates the effects of BC diet supplementation on luminal and mucosa-associated microbiota in the jejunum, caecum, and colon of rabbits. Twenty-one New Zealand White female rabbits were divided into three experimental groups (n = 7) receiving a commercial feed (CTRL group) and the same diet supplemented with 2.5% and 5% BC (2.5% BC and 5% BC groups, respectively), from 35 (weaning) to 90 days of age (slaughtering). At slaughter, the digestive tract was removed from each animal, then both content and mucosa-associated microbiota of jejunum, caecum, and colon were collected and analysed by Next Generation 16SrRNA Gene Sequencing. Significant differences were found in the microbial composition of the three groups (i.e., beta-diversity: p < 0.01), especially in the caecum and colon of the 2.5% BC group. The relative abundance analysis showed that the families most affected by the BC administration were Clostridia UCG-014, Barnesiellaceae, and Eggerthellaceae. A trend was also found for Lachnospiraceae, Akkermansiaceae, and Bacteroidaceae. A functional prediction has revealed several altered pathways in BC groups, with particular reference to amino acids and lactose metabolism. Firmicutes:Bacteroidetes ratio decreased in caecum luminal samples of the 2.5% BC group. These findings suggest that BC supplementation could positively affect the intestinal microbiota. However, further research is needed to establish the optimal administration dose.

PSXV-17 Effects of Dietary Supplementation with Spray Dried Plasma, Dried Intestinal Hydrolysates or Bovine Colostrum on the Growth and Intestinal Health of Weanling Pigs

Abstract In this study, we evaluated the potential of supplementing weaning diets with spray dried porcine plasma, dried porcine intestinal hydrolysates or bovine colostrum to modulate piglet growth, intestinal morphology, antioxidant status, and immunity. At weaning, 96 piglets were housed three piglets per pen in 32 pens. Each pen was assigned to one of the following dietary treatments: 1) basal diet (CTRL); 2) basal diet containing spray dried porcine plasma at 50 g/kg feed (PP); 3) basal diet containing dried porcine intestinal hydrolysates at 50 g/kg feed (PIH); 4) basal diet containing bovine colostrum at 50 g/kg feed (BC). At d 0 and 14, piglets were weighed and feed intake was daily recorded. Eight pigs per treatment (one per pen) were euthanized at d 14 and intestinal samples were collected to evaluate the antioxidant status, intestinal morphology and concentration of TNFα, interleukin (IL) 4, IL17, occludin, caspase 3 and proliferating cell nuclear antigen. Our results showed that PP and BC supplementation increased the final weight and G:F ratio compared with the other dietary treatments, whereas the ADG was greater in the BC group compared with PIH animals (P&amp;lt; 0.05). Furthermore, feeding pigs with the PP diet partially affected the intestinal antioxidant status, as evidenced by the increased concentration of glutathione peroxidase compared with the BC group (P&amp;lt; 0.05). BC supplementation also increased intestinal villus height compared with CTRL and PIH animals, as well as villus to crypt ratio compared with the CTRL group (P&amp;lt; 0.05). No other dietary effect on immunity or intestinal morphology was recorded. This study highlights the potential of dietary supplementation, such as plasma proteins and bovine colostrum, to act as a valid alternative to in-feed antibiotics, to reduce effect of weaning stress and maintain gut health and performance in in the post-weaning period.

β-carotene regulates cancer stemness in colon cancer in vivo and in vitro.

BACKGROUND/OBJECTIVESColorectal cancer (CRC) is the third most common cancer worldwide and has a high recurrence rate, which is associated with cancer stem cells (CSCs). β-carotene (BC) possesses antioxidant activity and several anticancer mechanisms. However, no investigation has examined its effect on colon cancer stemness.MATERIALS/METHODSCD133+CD44+ HCT116 and CD133+CD44+ HT-29 cells were isolated and analyzed their self-renewal capacity by clonogenic and sphere formation assays. Expressions of several CSCs markers and Wnt/β-catenin signaling were examined. In addition, CD133+CD44+ HCT116 cells were subcutaneously injected in xenograft mice and analyzed the effect of BC on tumor formation, tumor volume, and CSCs markers in tumors.RESULTSBC inhibited self-renewal capacity and CSC markers, including CD44, CD133, ALDH1A1, NOTCH1, Sox2, and β-catenin in vitro. The effects of BC on CSC markers were confirmed in primary cells isolated from human CRC tumors. BC supplementation decreased the number and size of tumors and delayed the tumor-onset time in xenograft mice injected with CD133+CD44+ HCT116 cells. The inhibitory effect of BC on CSC markers and the Wnt/β-catenin signaling pathway in tumors was confirmed in vivo as well.CONCLUSIONSThese results suggest that BC may be a potential therapeutic agent for colon cancer by targeting colon CSCs.

Spore-forming Bacillus coagulans SNZ 1969 improved intestinal motility and constipation perception mediated by microbial alterations in healthy adults with mild intermittent constipation: A randomized controlled trial

The spore-forming Bacillus coagulans has attracted attention for their therapeutic action in the colon. However, the mechanism of this action remains unclear. In this study, healthy subjects with mild intermittent constipation were supplemented with B. coagulans SNZ 1969 (BC) or the placebo for 8 weeks (n = 80). Then, we assessed colonic transit time (CTT), weekly complete spontaneous bowel movement (CSBM) scores, bowel discomfort symptom (BDS) scores, and 16S rRNA fecal microbiome profiles. The association between the critically altered gut microbiome and clinical outcomes was analyzed using redundancy analysis (RDA) and validated by receiver operating characteristic (ROC) curves. BC supplementation significantly improved CTT (p = 0.031), CSBM at weeks 2 (p = 0.045) and 9 (p = 0.038), and BDS at weeks 3 (p = 0.019) and 6 (p = 0.029) compared with the placebo, while altering the community composition of the gut microbiota. We also confirmed that BC was effectively delivered to the gut. Finally, the multivariate redundancy analysis concluded that BC-induced enrichment of Lactobacillales and diminishment of Synergistales were related to CTT improvements. This study provides important new data on how spore-forming B. coagulans SNZ 1969 contributes to improving gut motility and presents evidence supporting the use of B. coagulans SNZ 1969 in adults with mild intermittent constipation and habitual low intake of fruit and vegetables.

Low Dose of β-Carotene Regulates Inflammation, Reduces Caspase Signaling, and Correlates with Autophagy Activation in Cardiomyoblast Cell Lines.

BackgroundExcessive reactive oxygen species (ROS) stimulate mitochondrial damage that causes degenerative diseases such as cardiovascular disease (CVD). β-carotene (BC), a natural antioxidant able to counteract free radicals, acts as a cytoprotective agent. However, knowledge of the role of BC on cardiomyoblasts is limited. In this study, we explored its role on COX4, Tom20, Nfr1, Nrf2, Nf-κB, LC3, p62, caspase 3, and caspase 9 and its association with cardiomyoblast viability and survival.Material/MethodsH9C2 cell lines were seeded, cultivated until 90% to 100% confluency, and treated with various doses of BC: 10 μM, 1 μM, 0.1 μM, and 0.01 μM. After 24 h, the cells were harvested, lyzed, and tested for specific related protein expressions from each dose.ResultsLow-dose BC induced autophagy most effectively at 1 μM, 0.1 μM, and 0.01 μM, as indicated by a decrease of LC3II and p62 levels. We observed that Nf-κB protein levels were suppressed; Nrf2 was stimulated, but Nrf1 was not altered significantly. Further, low-dose BC might stimulate cell viability by reducing apoptotic signals of caspase 3 and 9. Notably, low-dose BC also showed potential to increase Tom20 protein levels.ConclusionsLow-dose BC supplementation shows beneficial effects, especially at 0.01 μM, by reducing inflammation through the suppression of Nf-κB and increase of Nrf2 level. Autophagy as a cellular maintenance mechanism was also stimulated, and the amount of the mitochondria marker Tom20 increased. Taken together, results showed that specific low-dose BC is effective and might improve cell viability by stimulating autophagy, inhibiting proinflammatory factors, and suppressing apoptosis.

β-Carotene exerts anti-colon cancer effects by regulating M2 macrophages and activated fibroblasts

The tumor microenvironment (TME), consisting of stromal fibroblasts, immune cells, cancer cells and other cell types, plays a crucial role in cancer progression and metastasis. M2 macrophages and activated fibroblasts (AFs) modulate behavior of cancer cells in the TME. Since nutritional effects on cancer progression, including colorectal cancer (CRC), may be mediated by alterations in the TME, we determined the ability of β-carotene (BC) to mediate anti-cancer effects through regulation of macrophage polarization and fibroblast activation in CRC. The M2 macrophage phenotype was induced by treating U937 cells with phorbol-12-myristate-13-acetate and interleukin (IL)-4. Treatment of these M2 macrophages with BC led to suppression of M2-type macrophage-associated markers and of the IL-6/STAT3 signaling pathway. In separate experiments, AFs were induced by treating CCD-18Co cells with transforming growth factor-β1. BC treatment suppressed expression of fibroblast activation markers. In addition, conditioned media from BC-treated M2 macrophages and AF inhibited cancer stem cell markers, colon cancer cell invasiveness and migration, and the epithelial-mesenchymal transition (EMT). In vivo, BC supplementation inhibited tumor formation and the expression of M2 macrophage markers in an azoxymethane/dextran sodium sulfate-induced colitis-associated CRC mouse model. To our knowledge, the present findings provide the first evidence suggesting that the potential therapeutic effects of BC on CRC are mediated by the inhibition of M2 macrophage polarization and fibroblast activation.

Abstract 1582: Catechol-O-methyltransferase (COMT) genotypes modify the effects of beta-carotene supplementation on cancer incidence in a controlled trial: findings from the ATBC Study

Abstract Large cancer prevention trials of vitamin supplementation have yielded mixed results over the past three decades. Whether and how genetic variation that influences oxidative stress or vitamin absorption, transport, and metabolism can modify responses to supplementation remains to be elucidated. Catechol-O-methyltransferase (COMT) genotypes have been shown to impact the effects of vitamin E (i.e., alpha-tocopherol) supplementation on cancer, and the present pharmacogenetic analysis examines interactions among COMT, beta-carotene supplements, and risk of cancer. In a case-control study nested within the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study of 29,133 Finnish men, we examined overall cancer risk based on the randomized assignment to beta-carotene supplementation (BC; 20 mg daily for 5-8 years) and two common genetic variants in COMT, rs4680 and rs4818 (genotyped by Illumina scans). Based on 2396 cases and 2235 non-cases, Cox proportional hazards models that adjusted for age, smoking intensity and duration, and randomized assignment to the trial vitamin E supplement were conducted to estimate overall cancer risk for the trial and 10-year post-trial periods combined. For rs4680, BC supplementation (versus no BC) was associated with higher cancer risk only in men with the AA (met/met) genotype (HR 1.29, 95% CI 1.08-1.53, P=0.004) as compared with the AG (met/val) and GG (val/val) genotypes (respectively, HR 1.04, 95% CI 0.93-1.16, P=0.52; and, HR 0.99, 95% CI 0.86-1.15, P=0.94). We observed a somewhat similar pattern for rs4818, with the BC supplement being associated with higher cancer risk in men with the GG and GC genotypes (respectively, HR 1.21, 95% CI 0.94-1.55, P=0.14; and, HR 1.15, 95% CI 1.01-1.30, P=0.03), but not the CC genotype (HR 0.99, 95% CI 0.88-1.11, P=0.80). Our findings indicate that the AA (met/met) genotype of COMT rs4680 and the G allele of rs4818 potentiate the increased cancer risk resulting from BC supplementation. Citation Format: Stephanie J. Weinstein, Jiaqi Huang, Kathryn Tayo Hall, Howard D. Sesso, Demetrius Albanes. Catechol-O-methyltransferase (COMT) genotypes modify the effects of beta-carotene supplementation on cancer incidence in a controlled trial: findings from the ATBC Study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1582.

β-carotene Suppresses Colon Cancer by Regulating M2 Macrophages and Tumor-associated Fibroblasts in Vitro and in Vivo (P02-015-19)

ObjectivesTumor-associated macrophages (TAMs) and tumor-associated fibroblasts (TAFs) are consisted of tumor microenvironment (TME), which are involved in cancer progression and metastasis. Interactions within TME induce M2 macrophage phenotype, TAMs, and activate TAFs. β-carotene (BC) is a well-known antioxidant and showed protective effects on several diseases, including cancers. The object of this study is to investigate the anti-colorectal cancer (CRC) effects of BC by controlling macrophage polarization and fibroblast activation. MethodsTAMs were induced by treating with phorbol-12-myristate-13-acetate (PMA) and interleukin-4 (IL-4) in U937 cells and TAFs were induced by treating with transforming growth factor-β1 (TGF-β1) in CCD-18Co cells. To understand the effect of TME on cancer cells, HCT116 colon cancer cells were co-cultured with TAM or TAF conditioned media. The effects of BC on the expressions of cancer stem cells (CSCs) markers, epithelial-mesenchymal transition (EMT) markers along with invasion and migration were investigated. To confirm these results, the azoxymethane (AOM) and dextran sodium sulfate (DSS)-induced colitis-associated CRC mice model was used. ResultsBC decreased M2 macrophage polarization with activating IL-6/STAT3 signaling pathways and suppressed the expressions of fibroblast activation markers and EMT markers. In addition, BC inhibited the expressions of TME-induced CSCs markers and EMT and suppressed cell invasion and migration. Furthermore, BC supplementation suppressed tumorigenesis and the expressions of M2 macrophage-associated markers, including CD206, Arg1, and Ym-1 as well as CSCs markers in vivo. ConclusionsBC suppressed CRC by regulating TAMs and TAFs in vitro and in vivo, which indicated the potential therapeutic effects of BC on inflammatory diseases. Funding SourcesThis research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education and Brain Korea 21 Plus.

Maternal Supplementation with β-Carotene During Pregnancy Disturbs Lipid Metabolism and Glucose Homoeostasis in F1 Female Mice.

β-Carotene (BC), a substitute for vitamin A, is widely used for its benefits. The present study investigates whether in-utero BC administration can alter lipid and glucose homoeostasis in offspring. Pregnant mice are supplemented with BC (1 mg kg-1 weight) by oral gavage once every 3 days, for a total of six doses. Increased visceral fat may be caused by up-regulated PPARγ (peroxisome proliferator-activated receptor gamma) and RXRα/β (retinoid X receptors) in liver and adipose tissue, and glucose intolerance is observed in F1 adult females prenatally supplemented with BC, while F1 males do not exhibit these symptoms. In females, increased serum leptin, resistin, and IL-6 and reduced adiponectin, caused by visceral obesity, may result in downregulated insulin receptor signaling in muscle and further account for glucose intolerance. Increased pancreatic β-cell mass might compensate for the downregulated insulin gene (ins2). Increased glucagon and α-cell mass, accompanied by upregulated glucagon gene (gcg), might also be risk factors for the development of diabetes. Maternal supplementation with BC disturbs lipid metabolism and induces glucose intolerance in F1 female mice, suggesting that BC supplementation during pregnancy should be used with caution.

Abstract 2275: Serum retinol and risk of overall and site-specific cancer in the Alpha Tocopherol, Beta-Carotene Cancer Prevention Study

Abstract Retinol, one of the most biologically active forms of vitamin A, influences many biologic pathways potentially related to cancer. However, results of observational studies of serum retinol and cancer risk have been mixed. We prospectively examined serum retinol and risk of overall and site-specific cancer in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study, a randomized, double-blind, placebo controlled trial of α-tocopherol (AT) and β-carotene (BC) supplementation conducted in male smokers in southwestern Finland. At enrollment, overnight fasting blood samples were collected and stored at -70°C protected from light. Serum retinol concentration was measured for all 29,133 participants using reverse-phase high performance liquid chromatography. 10,798 cancer cases occurred through 12/31/2012. Cox proportional hazards models were used to estimate the prospective association between quintiles of baseline serum retinol and overall and site-specific cancer incidence. Known or hypothesized risk factors for cancer were included in the multivariable model (AT and BC supplementation group, cigarettes smoked/day, years smoked, age, BMI, alcohol intake, and serum AT, BC, and cholesterol). Stratified analyses for all subgroups of the covariates (by category or by medians for continuous variables) and follow-up time to diagnosis (&amp;lt;10 vs. ≥10 y) were also performed for overall cancer and for the cancers for which an association with serum retinol was observed. After adjustment for age, higher serum retinol was associated with a lower risk of overall cancer (Q5 vs. Q1: HR=0.93, 95%CI=0.88-0.99, p-trend = 0.04). This finding was attenuated and no longer statistically significant after multivariable adjustment (MV-adj) (Q5 vs. Q1: MV-adj HR= 0.97, 95% CI = 0.91-1.03, p-trend = 0.43). Higher retinol concentrations were associated with increased risk of prostate cancer (Q5 vs. Q1: MV-adj HR=1.28, 95%CI = 1.13-1.45, p-trend &amp;lt; 0.0001) and lower risk of both lung and liver cancers (Q5 vs. Q1: liver MV-adj HR = 0.70, 95%CI = 0.50-0.99,p-trend = 0.03; lung MV-adj HR = 0.80, 95%CI = 0.72-0.88, p-trend &amp;lt; 0.0001). No associations with other cancers were observed. The inverse association between retinol and lung cancer was significantly modified by serum BC with a stronger association among men with higher serum BC (p for interaction = 0.018). No other statistically significant interactions were observed. In this large prospective study, higher serum retinol was associated with increased risk of prostate cancer and lower risk of lung and liver cancers. Understanding the biological mechanisms that underlie these associations may provide insight into retinol’s role in cancer prevention. Citation Format: Manila Hada, Alison Mondul, Stephanie Weinstein, Demetrius Albanes. Serum retinol and risk of overall and site-specific cancer in the Alpha Tocopherol, Beta-Carotene Cancer Prevention Study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2275. doi:10.1158/1538-7445.AM2017-2275

The effect of a negative energy balance status on β-carotene availability in serum and follicular fluid of nonlactating dairy cows

Maternal metabolic pressure due to a cow’s negative energy balance (NEB) has a negative effect on oocyte quality as a result of increased oxidative stress. In this study, we hypothesized that a NEB status may negatively affect the availability of β-carotene (bC, an antioxidant) in the micro-environment of the oocyte or follicular fluid (FF) and that daily bC supplementation can increase bC availability. We aimed to (1) determine the effect of a nutritionally induced NEB on bC concentrations in serum and FF as well as on the presence of bC metabolites, oxidative stress levels, and follicular growth in a nonlactating dairy cow model, and (2) investigate how this effect could be altered by dietary bC supplementation. Six multiparous nonlactating Holstein Friesian cows were subjected to 4 consecutive dietary treatments, 28 d each: (1) 1.2 × maintenance (M) or positive energy balance (PEB) without bC supplement (PEB-bC), (2) 1.2 × M with daily supplement of 2,000mg of bC comparable to the level of bC intake at grazing (PEB+bC), (3) 0.6 × M with 2,000mg of bC (NEB+bC), and (4) 0.6 × M (NEB-bC). At the end of each treatment, estrous cycles were synchronized and blood and FF of the largest follicle were sampled and analyzed for bC, retinol, α-tocopherol, free fatty acids, estradiol, and progesterone. Serum cholesterol, triglycerides, urea, insulin growth factor 1, growth hormone, total antioxidant status (TAS), and red blood cell glutathione (GSH) concentrations were determined as well. All cows lost body weight during both energy restriction periods and showed increased serum free fatty acid concentrations, illustrating a NEB. A dietary induced NEB reduced FF bC, but not plasma bC or plasma and FF retinol concentrations. However, bC and retinol concentrations drastically increased in both fluid compartments after bC supplementation. Follicular diameter was increased in supplemented PEB cows. Energy restriction reduced the TAS and red blood cell GSH, whereas daily bC supplementation could restore GSH concentrations, but not the TAS, to levels present in healthy PEB cows. In conclusion, daily bC supplementation can substantially improve bC and retinol availability in the oocyte’s micro-environment, irrespective of the energy balance, which may affect follicular development and oocyte quality in the presence of maternal metabolic stress. This knowledge can be of importance to optimize nutritional strategies in the dairy industry to feed for optimal oocyte quality and fertility.

Supplementation with β-carotene or vitamin E protects against increase in anaphylactic response in β-lactoglobulin-sensitized Balb/c mice: ex vivo study

Balb/c mice fed 0 mg, 20 mg of β-carotene (BC) or 50 mg of vitamin E (Vit E) per Kg of diet for 7 weeks were immunized intraperitoneally with β-Lactoglobulin (β-Lg). Specific IgG and IgE anti β-Lg were determined in the mouse sera by an enzyme-linked immunosorbent assay (ELISA), and the jejunum was used for assessment of local anaphylactic responses ex vivo in Ussing chambers. Short-circuit current (Isc), a measure of active ion transport, and epithelial conductance (G), a measure of passive ion permeability, were measured for each tissue. Specimens were also processed for examination by histological analysis. Compared with sensitized mice (unsupplemented mice), those fed BC or Vit E had lower β-Lg-specific IgG (p < 0.001 and p < 0.0001, respectively) and IgE levels (p < 0.02 and p < 0.01, respectively). A secretory response reflecting local intestinal anaphylaxis was evident in sensitized mice, as indicated by an increase in Isc (p < 0.009). Sensitized mice demonstrated also an enhanced epithelial permeability as measured by conductance (p < 0.03). BC supplementation resulted in a significant decrease in secretory response (p < 0.02) with no effect on the epithelial permeability. Interestingly, the secretory response and epithelial permeability were significantly reduced by supplementation with Vit E (p < 0.02 and p < 0.009, respectively). These findings provide evidence for the first time that both BC and Vit E regulate epithelial ion transport, but it should be noted that only Vit E-supplemented mice showed a decreased tissue conductance, indicating protection from β-Lg-induced epithelial permeability increase. Analysis of histological sections revealed that BC or Vit E treatment clearly reduced the microscopic lesions caused by β-Lg sensitization.

β-Carotene inhibits neuroblastoma tumorigenesis by regulating cell differentiation and cancer cell stemness

Neuroblastoma (NB) is the most common extracranial solid cancer in young children and malignant NB cells have been shown to possess cancer stem cell (CSC) characteristics. Thus, the successful elimination of CSCs represents a strategy for developing an effective preventive and chemotherapeutic agent. CSCs are characterized by differentiation and tumorigenicity. β-Carotene (BC) has been associated with many anticancer mechanisms, although the efficacy of BC on CSCs remains unclear. In the present study, the effects of BC on tumor cell differentiation and tumorigenicity was investigated using a xenograft model. Mice were pretreated with BC for 21days, then received a subcutaneous injection of SK-N-BE(2)C cells. Both tumor incidence and tumor growth were significantly inhibited for mice that received BC supplementation compared to the control group. Treatment with BC has also been shown to induce tumor cell differentiation by up-regulating differentiation markers, such as vimentin, peripherin, and neurofilament. Conversely, BC treatment has been shown to significantly suppress tumor stemness by down-regulating CSC markers such as Oct 3/4 and DLK1. BC treatment also significantly down-regulated HIF1-α expression and its downstream target, vascular endothelial growth factor (VEGF). Taken together, these results suggest that BC is a potential chemotherapeutic reagent for the treatment of NB, and mediates this effect by regulating the differentiation and stemness of CSCs, respectively.

Embryonic phenotype, β-carotene and retinoid metabolism upon maternal supplementation of β-carotene in a mouse model of severe vitamin A deficiency

We investigated the effect of β-carotene (bC) supplementation during pregnancy in a mouse model of severe vitamin A deficiency, i.e. Lrat−/−Rbp−/− dams maintained on a vitamin A-deficient diet during gestation. bC, a provitamin A carotenoid, can be enzymatically cleaved to form vitamin A for use by the developing embryo. We found that an acute supplementation (13.5days post coitum, dpc) of bC to Lrat−/−Rbp−/− dams on a vitamin A-deficient diet activated transcriptional mechanisms in the developing tissues to maximize the utilization of bC provided to the dams. Nevertheless, these regulatory mechanisms are inefficient under this regimen, as the embryonic phenotype was not improved. We further investigated the effect of a repeated supplementation of bC during a crucial developmental period (6.5–9.5dpc) on the above-mentioned mouse model. This treatment improved the embryonic abnormalities, as 40% of the embryos showed a normal phenotype. In addition, analysis of retinoic acid-responsive genes, such as Cyp26a1 in these embryos suggests that bC cleavage results in the production of retinoic acid which then can be used by the embryo. Taken together, these in vivo studies show that bC can be used as a source of vitamin A for severely vitamin A-deficient mammalian embryos.

Role of LDL receptor in the uptake of beta‐carotene by murine placenta and the embryo

Beta‐carotene (bC) is the most abundant precursor of vitamin A in the human diet. Vitamin A derived retinoids are essential for normal embryonic development. Uptake of intact bC followed by local de novo biosynthesis of retinoids by cleavage of bC can be an important source of retinoids during embryonic development. Intact bC is circulated in association with various lipoprotein particles, especially apoE/apoB containing lipoproteins such as chylomicrons, VLDL and LDL.Potential role of LDL receptor (LDLr) in the uptake of supplemented bC by maternal and developing tissues was investigated. LDLr knockout female mice were mated with LDLr heterozygous males and vice versa to obtain embryos of the same litter that are either heterozygous or lack LDLr altogether. All mice were maintained on a regular chow diet throughout the lifespan. At 13.5 days of gestation, dams were intraperitoneally supplemented with a physiological dose of 20–50 μg/g body weight of bC. Maternal serum, liver, placenta and embryos were collected after 24 hours and analyzed by HPLC and qPCR.Hepatic bC levels were reduced in LDLr knockout dams. Placenta of heterozygous embryos had higher bC levels as compared to the knockout placenta, an effect observed only in case of LDLr knockout dams. Maternal and embryonic genotype did not have an effect on bC levels in embryos. Preliminary qPCR analysis of placenta revealed that both bC supplementation and LDLr genotype of the placenta had an effect on the mRNA expression of genes involved in lipid metabolism. These results suggest that LDLr mediates uptake of bC 1) in the liver and 2) in the placenta of dams lacking LDLr. In addition to this, the data suggested that a single dose of bC at mid‐gestation may alter genes involved in the lipid metabolism in the placenta and the embryo.Grant Funding Source: NIH

Does beta‐carotene‐9′,10′‐oxygenase (CMO2) generate retinoic acid during embryonic development?

Vitamin A is an essential nutrient throughout the mammalian lifecycle, especially for normal embryonic development. Most of the world relies on β‐carotene (bC), a provitamin A carotenoid, as its main vitamin A source. bC is converted to vitamin A by one of two enzymatic cleavage mechanisms: central cleavage by β‐ carotene‐15,15′‐oxygenase (CMO1), or asymmetric cleavage by β‐ carotene‐9′,10′‐oxygenase (CMO2). ~95% of bC conversion to vitamin A is attributed to CMO1, while CMO2, a mitochondrial enzyme, is thought to contribute minimally to vitamin A production in vivo. Both CMO1 and CMO2 are expressed in the developing tissues, and local bC cleavage by embryonic CMO1 can generate adequate vitamin A for normal development. When CMO1−/−RBP−/− dams were fed a vitamin A‐deficient diet during pregnancy, embryos developed gross malformations including eye defects, peripheral edema, and exencephaly. Supplementation of CMO1−/−RBP−/− dams with bC from 6.5–9.5 days post coitum partly improved the vitamin A deficiency symptoms of the double knockout embryos, despite the lack of CMO1. To determine whether bC cleavage by CMO2 contributed to this improvement, triple knockout mice (CMO1−/−RBP−/− CMO2−/−) were also generated, and analyzed under the same dietary regimen. The frequency of defects of CMO1−/−RBP−/−CMO2−/− embryos from dams fed a vitamin A‐deficient diet was similar to that observed in double‐knockout embryos. Experiments are ongoing to investigate whether the embryonic defects of triple knockout embryos can be improved by bC supplementation from 6.5–9.5 dpc. These experiments will shed more light on the role of CMO2 as a potential retinoic acid‐generating enzyme during embryonic development.Grant Funding Source: NIH; Gates Foundation