The mevalonate suppressor δ-tocotrienol increases AMPA receptor-mediated neurotransmission.

Synaptic dysfunction is a hallmark of aging and is found in several neurological disorders such as Alzheimer's disease. A common mechanism related to synaptic dysfunction is dysregulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which mediate excitatory neurotransmission and synaptic plasticity. Accumulating evidence suggests that tocotrienols, vitamin E molecules that contain an isoprenoid side chain, may promote cognitive improvement in hippocampal-dependent learning tasks. Tocotrienols have also been shown to reduce the secretion of ß-amyloid (Aß) and cholesterol biosynthesis in part by downregulating 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme that controls flux of the mevalonate pathway and cholesterol biosynthesis. We hypothesized that tocotrienols might promote cognitive improvement by increasing AMPA receptor-mediated synaptic transmission. Here, we found that δ-tocotrienol increased surface levels of GluA1 but not the GluA2 AMPA receptor subunit in primary hippocampal neurons. Unexpectedly, δ-tocotrienol treatment caused a decrease in the phosphorylation of GluA1 at Serine 845 with no significant changes in GluA1 at Serine 831. Moreover, δ-tocotrienol increased spontaneous excitatory postsynaptic current (sEPSC) amplitude and reduced the secretion of Aß40 in primary hippocampal neurons. Taken together, our findings suggest that δ-tocotrienol increases AMPA receptor-mediated neurotransmission via noncanonical changes in GluA1 phosphorylation status. These findings suggest that δ-tocotrienol may be beneficial in ameliorating synaptic dysfunction found in aging and neurological disease.

Tocotrienols: Dietary Supplements for Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is one of the leading causes of death worldwide. Emphysema and chronic bronchitis are the two major phenotypes of COPD, which have many symptoms, such as dyspnea, chronic cough, and mucus overproduction. Emphysema is characterized by the destruction of the alveolar wall, while chronic bronchitis is characterized by limitations in expiratory airflow. Cigarette smoking is the most significant risk factor for the pathogenesis of COPD in the developed world. Chronic inflammation contributes to the onset and progression of the disease and furthers the risk of comorbidities. Current treatment options and prevention strategies for COPD are very limited. Tocotrienols are a group of vitamin E molecules with antioxidant and anti-inflammatory properties. Individual tocotrienols (α, γ, and δ) have shown their ability to attenuate inflammation specifically via suppressing nuclear factor-κB-mediated cytokine production. The δ- and γ-forms of tocotrienols have been indicated as the most effective in the prevention of macrophage infiltration, production of reactive oxygen species, and cytokine secretion. This review briefly discusses the pathogenesis of COPD and the role of inflammation therein. Furthermore, we summarize the in vitro and in vivo evidence for the anti-inflammatory activity of tocotrienols and their potential application to COPD management. Coupled with the bioavailability and safety profile of tocotrienols, the ability of these compounds to modulate COPD progression by targeting the inflammation pathways renders them potential candidates for novel therapeutic approaches in the treatment of COPD patients.

Dietary Annatto-Extracted Tocotrienol Reduces Inflammation and Oxidative Stress, and Improves Macronutrient Metabolism in Obese Mice: A Metabolic Profiling Study.

Obesity and its related complications are a world-wide health problem. Dietary tocotrienols (TT) have been shown to improve obesity-associated metabolic disorders, such as hypercholesterolemia, hyperglycemia, and gut dysbiosis. This study examined the hypothesis that the antioxidant capacity of TT alters metabolites of oxidative stress and improves systemic metabolism. C57BL/6J mice were fed either a high-fat diet (HFD control) or HFD supplemented with 800 mg annatto-extracted TT/kg (HFD+TT800) for 14 weeks. Sera from obese mice were examined by non-targeted metabolite analysis using UHPLC/MS. Compared to the HFD group, the HFD+TT800 group had higher levels of serum metabolites, essential amino acids (lysine and methionine), sphingomyelins, phosphatidylcholine, lysophospholipids, and vitamins (pantothenate, pyridoxamine, pyridoxal, and retinol). TT-treated mice had lowered levels of serum metabolites, dicarboxylic fatty acids, and inflammatory/oxidative stress markers (trimethylamine N-oxide, kynurenate, 12,13-DiHOME, and 13-HODE + 9-HODE) compared to the control. The results suggest that TT supplementation lowered inflammation and oxidative stress (oxidized glutathione and GSH/GSSH) and improved macronutrient metabolism (carbohydrates) in obese mice. Thus, TT actions on metabolites were beneficial in reducing obesity-associated hypercholesterolemia/hyperglycemia. The effects of a non-toxic dose of TT in mice support the potential for clinical applications in obesity and metabolic disease.

Tocotrienol Supplementation Led to Higher Serum Levels of Lysophospholipids but Lower Acylcarnitines in Postmenopausal Women: A Randomized Double-Blinded Placebo-Controlled Clinical Trial.

Osteoporosis is a major health problem in postmenopausal women. Herein we evaluated the effects of 12-week tocotrienols (TT) supplementation on serum metabolites in postmenopausal, osteopenic women. Eighty-nine participants (59.7 ± 6.8 yr, BMI 28.7 ± 5.7 kg/m2) were assigned to 3 treatments: placebo (860 mg olive oil/day), 300mg TT (300 mg TT/day), and 600mg TT (600 mg TT/day) for 12 weeks. TT consisted of 90% δ-TT and 10% γ-TT. In this metabolomic study, we evaluated the placebo and 600mgTT at baseline and 12 weeks. As expected, TT and its metabolite levels were higher in the supplemented group after 12 weeks. At baseline, there were no differences in demographic parameters or comprehensive metabolic panels (CMP). Metabolomics analysis of serum samples revealed that 48 biochemicals were higher and 65 were lower in the 600mg TT group at 12 weeks, compared to baseline. The results confirmed higher serum levels of tocotrienols and lysophospholipids, but lower acylcarnitines and catabolites of tryptophan and steroids in subjects given 600mg TT. In summary, 12-week TT supplementation altered many serum metabolite levels in postmenopausal women. The present study supports our previous findings that TT supplementation helps reduce bone loss in postmenopausal osteopenic women by suppressing inflammation and oxidative stress. Furthermore, the body incorporates TT which restructures biomembranes and modifies phospholipid metabolism, a response potentially linked to reduced inflammation and oxidative stress.

Synergistic Impact of Xanthorrhizol and d-δ-Tocotrienol on the Proliferation of Murine B16 Melanoma Cells and Human DU145 Prostate Carcinoma Cells.

Isoprenoids suppress the mevalonate pathway that provides prenyl groups for the posttranslational modification of growth-regulating proteins. We hypothesize that xanthorrhizol and d-δ-tocotrienol synergistically suppress the growth of murine B16 melanoma and human DU145 prostate carcinoma cells. Xanthorrhizol (0-200 µmol/L; half maximal inhibitory concentration [IC50] = 65 µmol/L) and d-δ-tocotrienol (0-40 µmol/L; IC50 = 20 µmol/L) each induced a concentration-dependent suppression of the proliferation of B16 cells and concurrent cell cycle arrest at the G1 phase. A blend of 16.25 µmol/L xanthorrhizol and 10 µmol/L d-δ-tocotrienol suppressed B16 cell proliferation by 69%, an impact greater than the sum of those induced by xanthorrhizol (15%) and d-δ-tocotrienol (12%) individually. The blend cumulatively reduced the levels of cyclin-dependent kinase four and cyclin D1, key regulators of cell cycle progression at the G1 phase. The expression of RAS and extracellular signal-regulated kinase (ERK1/2) in the proliferation-stimulating RAS-RAF-MEK-ERK pathway was downregulated by the blend. Xanthorrhizol also induced a concentration-dependent suppression of the proliferation of DU145 cells with concomitant morphological changes. Isobologram confirmed the synergistic effect of xanthorrhizol and d-δ-tocotrienol on DU145 cell proliferation with combination index values ranging 0.61-0.94. Novel combinations of isoprenoids with synergistic actions may offer effective approaches in cancer prevention and therapy.

Annatto-extracted tocotrienols improve glucose homeostasis and bone properties in high-fat diet-induced type 2 diabetic mice by decreasing the inflammatory response.

Diabetes is a risk factor for osteoporosis. Annatto-extracted tocotrienols (TT) have proven benefits in preserving bone matrix. Here, we evaluated the effects of dietary TT on glucose homeostasis, bone properties, and liver pro-inflammatory mRNA expression in high-fat diet (HFD)-induced type 2 diabetic (T2DM) mice. 58 male C57BL/6 J mice were divided into 5 groups: low-fat diet (LFD), HFD, HFD + 400 mgTT/kg diet (T400), HFD + 1600 mgTT/kg diet (T1600), and HFD + 200 mg metformin/kg (Met) for 14 weeks. Relative to the HFD group, both TT-supplemented groups (1) improved glucose homeostasis by lowering the area under the curve for both glucose tolerance and insulin tolerance tests, (2) increased serum procollagen I intact N-terminal propeptide (bone formation) level, trabecular bone volume/total volume, trabecular number, connectivity density, and cortical thickness, (3) decreased collagen type 1 cross-linked C-telopeptide (bone resorption) levels, trabecular separation, and structure model index, and (4) suppressed liver mRNA levels of inflammation markers including IL-2, IL-23, IFN-γ, MCP-1, TNF-α, ITGAX and F4/80. There were no differences in glucose homeostasis and liver mRNA expression among T400, T1600, and Met. The order of osteo-protective effects was LFD ≥T1600 ≥T400 = Met >HFD. Collectively, these data suggest that TT exerts osteo-protective effects in T2DM mice by regulating glucose homeostasis and suppressing inflammation.

A 12-week evaluation of annatto tocotrienol supplementation for postmenopausal women: safety, quality of life, body composition, physical activity, and nutrient intake.

BACKGROUND: Evidence suggests that tocotrienols may benefit bone health in osteopenic women. However, their safety in this population has never been investigated. This study was to evaluate the safety of a 12-week supplementation of annato tocotrienol in postmenopausal osteopenic women, along with effects of the supplementation on quality of life, body composition, physical activity, and nutrient intake in this population. METHODS: Eighty nine postmenopausal osteopenic women were randomly assigned to 3 treatment arms: (1) Placebo (430 mg olive oil/day), (2) Low tocotrientol (Low TT) (430 mg tocotrienol/day from DeltaGold 70 containing 300 mg tocotrienol) and (3) High tocotrienol (High TT) (860 mg tocotrienol/day from DeltaGold 70 containing 600 mg tocotrienol) for 12 weeks. DeltaGold 70 is an extract from annatto seed with 70% tocotrienol consisting of 90% delta-tocotrienol and 10% gamma-tocotrienol. Safety was examined by assessing liver enzymes (aspartate aminotransferase, alanine aminotransferase), alkaline phosphatase, bilirubin, kidney function (blood urea nitrogen and creatinine), electrolytes, glucose, protein, albumin, and globulin at 0, 6, and 12 weeks. Serum tocotrienol and tocopherol concentrations were assessed and pills counted at 0, 6, and 12 weeks. Quality of life, body composition, physical activity, and dietary macro- and micro-nutrient intake were evaluated at 0 and 12 weeks. A mixed model of repeated measures ANOVA was applied for analysis. RESULTS: Eighty seven subjects completed the study. Tocotrienol supplementation did not affect liver or kidney function parameters throughout the study. No adverse event due to treatments was reported by the participants. Tocotrienol supplementation for 6 weeks significantly increased serum delta-tocotrienol level and this high concentration was sustained to the end of study. There was no difference in serum delta-tocotrienol levels between the Low TT and the High TT groups. No effects of tocotrienol supplementation were observed on quality of life, body composition, physical activity, and nutrient intake. CONCLUSIONS: Annatto-derived tocotrienol up to 600 mg per day for 12 weeks appeared to be safe in postmenopausal osteopenic women, particularly in terms of liver and kidney functions. Tocotrienol supplementation for 12 weeks did not affect body composition, physical activity, quality of life, or intake of macro- and micro-nutrients in these subjects. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02058420 . TITLE: Tocotrienols and bone health of postmenopausal women.

Potential roles of vitamin E in age-related changes in skeletal muscle health.

Skeletal muscle disorders including sarcopenia are prevalent during the complex biological process of aging. Loss of muscle mass and strength commonly seen in sarcopenia is induced by impaired neuromuscular innervation, transition of skeletal muscle fiber type, and reduced muscle regenerative capacity, all attributable to chronic inflammation, oxidative stress, and mitochondrial dysfunction. Current literature suggests that vitamin E molecules (α-, ß-, γ-, δ-tocopherols and the corresponding tocotrienols) with their antioxidant and anti-inflammatory capabilities may mitigate age-associated skeletal dysfunction and enhance muscle regeneration, thus attenuating sarcopenia. Preclinical and human experimental studies show that vitamin E benefits myoblast proliferation, differentiation, survival, membrane repair, mitochondrial efficiency, muscle mass, muscle contractile properties, and exercise capacity. Limited number of human cross-sectional observational studies reveal positive associations between serum tocopherol level and muscle strength. Several factors, including difficulties in validating vitamin E intake and deficiency, variations in muscle-protective activity and metabolism of diverse forms of vitamin E, and lack of understanding of the mechanisms of action, preclude randomized clinical trials of vitamin E in people with sarcopenia. Future research should consider long-term clinical trials of with adequate sample size, advanced imaging technology and omics approaches to investigate underlying mechanisms and assess clinically meaningful parameters such as muscle strength, physical performance, and muscle mass in sarcopenia prevention and/or treatment.

The Potential of Isoprenoids in Adjuvant Cancer Therapy to Reduce Adverse Effects of Statins.

The mevalonate pathway provides sterols for membrane structure and nonsterol intermediates for the post-translational modification and membrane anchorage of growth-related proteins, including the Ras, Rac, and Rho GTPase family. Mevalonate-derived products are also essential for the Hedgehog pathway, steroid hormone signaling, and the nuclear localization of Yes-associated protein and transcriptional co-activator with PDZ-binding motif, all of which playing roles in tumorigenesis and cancer stem cell function. The phosphatidylinositol-4,5-bisphosphate 3-kinase-AKT-mammalian target of rapamycin complex 1 pathway, p53 with gain-of-function mutation, and oncoprotein MYC upregulate the mevalonate pathway, whereas adenosine monophosphate-activated protein kinase and tumor suppressor protein RB are the downregulators. The rate-limiting enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), is under a multivalent regulation. Sterol regulatory element binding protein 2 mediates the sterol-controlled transcriptional downregulation of HMGCR. UbiA prenyltransferase domain-containing protein-1 regulates the ubiquitination and proteasome-mediated degradation of HMGCR, which is accelerated by 24, 25-dihydrolanosterol and the diterpene geranylgeraniol. Statins, competitive inhibitors of HMGCR, deplete cells of mevalonate-derived intermediates and consequently inhibit cell proliferation and induce apoptosis. Clinical application of statins is marred by dose-limiting toxicities and mixed outcomes on cancer risk, survival and mortality, partially resulting from the statin-mediated compensatory upregulation of HMGCR and indiscriminate inhibition of HMGCR in normal and tumor cells. Tumor HMGCR is resistant to the sterol-mediated transcriptional control; consequently, HMGCR is upregulated in cancers derived from adrenal gland, blood and lymph, brain, breast, colon, connective tissue, embryo, esophagus, liver, lung, ovary, pancreas, prostate, skin, and stomach. Nevertheless, tumor HMGCR remains sensitive to isoprenoid-mediated degradation. Isoprenoids including monoterpenes (carvacrol, L-carvone, geraniol, perillyl alcohol), sesquiterpenes (cacalol, farnesol, ß-ionone), diterpene (geranylgeranyl acetone), "mixed" isoprenoids (tocotrienols), and their derivatives suppress the growth of tumor cells with little impact on non-malignant cells. In cancer cells derived from breast, colon, liver, mesothelium, prostate, pancreas, and skin, statins and isoprenoids, including tocotrienols, geraniol, limonene, ß-ionone and perillyl alcohol, synergistically suppress cell proliferation and associated signaling pathways. A blend of dietary lovastatin and δ-tocotrienol, each at no-effect doses, suppress the growth of implanted murine B16 melanomas in C57BL6 mice. Isoprenoids have potential as adjuvant agents to reduce the toxicities of statins in cancer prevention or therapy.

Tocotrienols for bone health: a translational approach.

Osteoporosis, a degenerative bone disease, is characterized by low bone mass and microstructural deterioration of bone tissue resulting in aggravated bone fragility and susceptibility to fractures. The trend of extended life expectancy is accompanied by a rise in the prevalence of osteoporosis and concomitant complications in the elderly population. Epidemiological evidence has shown an association between vitamin E consumption and the prevention of age-related bone loss in elderly women and men. Animal studies show that ingestion of vitamin E, especially tocotrienols, may benefit bone health in terms of maintaining higher bone mineral density and improving bone microstructure and quality. The beneficial effects of tocotrienols on bone health appear to be mediated via antioxidant/anti-inflammatory pathways and/or 3-hydroxy-3-methylglutaryl coenzyme A mechanisms. We discuss (1) an overview of the prevalence and etiology of osteoporosis, (2) types of vitamin E (tocopherols versus tocotrienols), (3) findings of tocotrienols and bone health from published in vitro and animal studies, (4) possible mechanisms involved in bone protection, and (5) challenges and future direction for research.

Synergistic Impact of d-δ-Tocotrienol and Geranylgeraniol on the Growth and HMG CoA Reductase of Human DU145 Prostate Carcinoma Cells.

The growth-suppressive effect of d-δ-tocotrienol and geranylgeraniol is at least partially attributed to their impact on 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, the rate-limiting enzyme in the mevalonate pathway that provides essential intermediates for the posttranslational modification of growth-related proteins including RAS. We hypothesize that these agents synergistically impact cell growth based on their complementary mechanisms of action with HMG CoA reductase. d-δ-tocotrienol (0-40 µmol/L; half maximal inhibitory concentration [IC50] = 15 µmol/L) and geranylgeraniol (0-100 µmol/L; IC50 = 60 µmol/L) each induced concentration-dependent suppression of the growth of human DU145 prostate carcinoma cells. Blends of the two agents synergistically suppressed the growth of DU145 cells, with combination index values ranging 0.67-0.75. While 7.5 µmol/L d-δ-tocotrienol and 30 µmol/L geranylgeraniol individually had no impact on cell cycle distribution in DU145 cells, a blend of the agents induced cell cycle arrest at the G1 phase. The synergistic downregulation of the expression of HMG CoA reductase by 7.5 µmol/L d-δ-tocotrienol and 30 µmol/L geranylgeraniol was accompanied by a reduction in membrane K-RAS protein. Our finding supports the cancer chemopreventive action of plant-based diets and their isoprenoid constituents. Properly formulated isoprenoids and derivatives may provide novel approaches in prostate cancer prevention and therapy.

Therapeutic properties of green tea against environmental insults.

Pesticides, smoke, mycotoxins, polychlorinated biphenyls (PCBs), and arsenic are the most common environmental toxins and toxicants to humans. These toxins and toxicants may impact on human health at the molecular (DNA, RNA, or protein), organelle (mitochondria, lysosome, or membranes), cellular (growth inhibition or cell death), tissue, organ, and systemic levels. Formation of reactive radicals, lipid peroxidation, inflammation, genotoxicity, hepatotoxicity, embryotoxicity, neurological alterations, apoptosis, and carcinogenic events are some of the mechanisms mediating the toxic effects of the environmental toxins and toxicants. Green tea, the nonoxidized and nonfermented form of tea that contains several polyphenols, including green tea catechins, exhibits protective effects against these environmental toxins and toxicants in preclinical studies and to a much-limited extent, in clinical trials. The protective effects are collectively mediated by antioxidant, antiinflammatory, antimutagenic, hepatoprotective and neuroprotective, and anticarcinogenic activities. In addition, green tea modulates signaling pathway including NF-κB and ERK pathways, preserves mitochondrial membrane potential, inhibits caspase-3 activity, down-regulates proapoptotic proteins, and induces the phase II detoxifying pathway. The bioavailability and metabolism of green tea and its protective effects against environmental insults induced by pesticides, smoke, mycotoxins, PCBs, and arsenic are reviewed in this paper. Future studies with emphasis on clinical trials should identify biomarkers of green tea intake, examine the mechanisms of action of green tea polyphenols, and investigate potential interactions of green tea with other toxicant-modulating dietary factors.

Safety and efficacy of tocotrienol supplementation for bone health in postmenopausal women: protocol for a dose-response double-blinded placebo-controlled randomised trial.

INTRODUCTION: Osteoporosis is a major health concern in postmenopausal women, and oxidative stress contributes to the development of bone loss. Cellular studies and ovariectomised rat model mimicking bone loss in postmenopausal women show the bone-protective effect of tocotrienols (TTs) with antioxidant capability. We aim to access the safety and efficacy of TT consumption for bone health in postmenopausal women. METHODS AND ANALYSIS: In this 12-week randomised double-blinded placebo-controlled trial for the effects of dietary TT supplementation in postmenopausal women, postmenopausal women aged 45 years and older with at least 1 year after menopause and bone mineral density T-score at the spine and/or hip 2.5 or more below the reference values will be randomly assigned to 3 daily supplements: (1) placebo group receiving 860 mg olive oil, (2) low TT group receiving 430 mg of 70% pure TTs (containing 300 mg TT) and (3) high TT group receiving 860 mg of 70% pure TTs (600 mg TT). The primary outcome measure will be urinary N-terminal telopeptide. The secondary outcome measures will be serum bone-specific alkaline phosphatase, receptor activator of nuclear factor-κB ligand, osteoprotegerin, urinary 8-hydroxy-2'-deoxyguanosine and quality of life. At 0, 6 and 12 weeks, the following will be assessed: (1) primary and secondary outcome measures; (2) serum TT and tocopherol concentrations; (3) physical activity and food frequency questionnaires. Liver function will be monitored every 6 weeks for safety. 'Intent-to-treat' principle will be employed for data analysis. A model of repeated measurements with random effect error terms will be applied. Analysis of covariance, χ2 analysis and regression will be used for comparisons. ETHICS AND DISSEMINATION: This study was approved by the Bioethics Committee of the Texas Tech University Health Sciences Center. The findings of this trial will be submitted to a peer-reviewed journal in the areas of bone or nutrition and international conferences. TRIAL REGISTRATION NUMBER: NCT02058420; results.

Peroxisome proliferator-activated receptor γ down-regulation mediates the inhibitory effect of d-δ-tocotrienol on the differentiation of murine 3T3-F442A preadipocytes.

Tocotrienols accelerate the degradation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase that catalyzes the biosynthesis of mevalonate; the latter is essential for preadipocyte differentiation. Tocotrienols also down-regulate peroxisome proliferator-activated receptor γ (PPARγ), a key regulator of adipocyte differentiation. We hypothesized that mevalonate deprivation and PPARγ down-regulation mediate d-δ-tocotrienol-induced inhibition of adipocyte differentiation. The objectives of this study were to determine the effect of d-δ-tocotrienol on 3T3-F442A preadipocyte differentiation and the involvement of PPARγ and mevalonate. Murine 3T3-F442A preadipocytes were incubated with d-δ-tocotrienol (2.5-10 µmol/L) for 8 days. AdipoRed assay and Oil Red O staining showed that d-δ-tocotrienol dose-dependently reduced the intracellular triglyceride content. Concomitantly, d-δ-tocotrienol dose-dependently inhibited glucose uptake by 3T3-F442A cells and the expression of GLUT4, HMG CoA reductase, and p-Akt proteins. The effects of d-δ-tocotrienol on intracellular triglyceride content and glucose uptake were attenuated by rosiglitazone, an agonist of PPARγ, but not supplemental mevalonate (100 µmol/L). In contrast, mevalonate, but not rosiglitazone, reversed the effects of lovastatin, a competitive inhibitor of HMG CoA reductase shown to inhibit adipocyte differentiation via mevalonate deprivation. Trypan blue staining revealed no changes in cell viability after a 48-hour incubation of 3T3-F442A cells with d-δ-tocotrienol (0-80 µmol/L), suggesting that the adipogenesis-suppressive activity of d-δ-tocotrienol was independent of cytotoxicity. In conclusion, these findings demonstrate the antiadipogenic effect of d-δ-tocotrienol via PPARγ down-regulation.

Potential of tocotrienols in the prevention and therapy of Alzheimer's disease.

Currently there is no cure for Alzheimer's disease (AD); clinical trials are underway to reduce amyloid generation and deposition, a neuropathological hallmark in brains of AD patients. While genetic factors and neuroinflammation contribute significantly to AD pathogenesis, whether increased cholesterol level is a causative factor or a result of AD is equivocal. Prenylation of proteins regulating neuronal functions requires mevalonate-derived farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). The observation that the levels of FPP and GGPP, but not that of cholesterol, are elevated in AD patients is consistent with the finding that statins, competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, reduce FPP and GGPP levels and amyloid ß protein production in preclinical studies. Retrospective studies show inverse correlations between incidence of AD and the intake and serum levels of the HMG CoA reductase-suppressive tocotrienols; tocopherols show mixed results. Tocotrienols, but not tocopherols, block the processing and nuclear localization of sterol regulatory element binding protein-2, the transcriptional factor for HMG CoA reductase and FPP synthase, and enhance the degradation of HMG CoA reductase. Consequently, tocotrienols deplete the pool of FPP and GGPP and potentially blunt prenylation-dependent AD pathogenesis. The antiinflammatory activity of tocotrienols further contributes to their protection against AD. The mevalonate- and inflammation-suppressive activities of tocotrienols may represent those of an estimated 23,000 mevalonate-derived plant secondary metabolites called isoprenoids, many of which are neuroprotective. Tocotrienol-containing plant foods and tocotrienol derivatives and formulations with enhanced bioavailability may offer a novel approach in AD prevention and treatment.

Inhibition of the STAT3 signaling pathway contributes to apigenin-mediated anti-metastatic effect in melanoma.

Signal transducer and activator of transcription 3 (STAT3) signaling is constantly activated in human melanoma, and promotes melanoma metastasis. The dietary flavonoid apigenin is a bioactive compound that possesses low toxicity and exerts anti-metastatic activity in melanoma. However, the anti-metastasis mechanism of apigenin has not been fully elucidated. In the present study, we showed that apigenin suppressed murine melanoma B16F10 cell lung metastasis in mice, and inhibited cell migration and invasion in human and murine melanoma cells. Further study indicated that apigenin effectively suppressed STAT3 phosphorylation, decreased STAT3 nuclear localization and inhibited STAT3 transcriptional activity. Apigenin also down-regulated STAT3 target genes MMP-2, MMP-9, VEGF and Twist1, which are involved in cell migration and invasion. More importantly, overexpression of STAT3 or Twist1 partially reversed apigenin-impaired cell migration and invasion. Our data not only reveal a novel anti-metastasis mechanism of apigenin but also support the notion that STAT3 is an attractive and promising target for melanoma treatment.

Trans, trans-farnesol as a mevalonate-derived inducer of murine 3T3-F442A pre-adipocyte differentiation.

Based on our finding that depletion of mevalonate-derived metabolites inhibits adipocyte differentiation, we hypothesize that trans, trans-farnesol (farnesol), a mevalonate-derived sesquiterpene, induces adipocyte differentiation. Farnesol dose-dependently (25-75 µmol/L) increased intracellular triglyceride content of murine 3T3-F442A pre-adipocytes measured by AdipoRed™ Assay and Oil Red-O staining. Concomitantly, farnesol dose-dependently increased glucose uptake and glucose transport protein 4 (GLUT4) expression without affecting cell viability. Furthermore, quantitative real-time polymerase chain reaction and Western blot showed that farnesol increased the mRNA and protein levels of peroxisome proliferator-activated receptor γ (PPARγ), a key regulator of adipocyte differentiation, and the mRNA levels of PPARγ-regulated fatty acid-binding protein 4 and adiponectin; in contrast, farnesol downregulated Pref-1 gene, a marker of pre-adipocytes. GW9662 (10 µmol/L), an antagonist of PPARγ, reversed the effects of farnesol on cellular lipid content, suggesting that PPARγ signaling pathway may mediate the farnesol effect. Farnesol (25-75 µmol/L) did not affect the mRNA level of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme in the mevalonate pathway. Farnesol may be the mevalonate-derived inducer of adipocyte differentiation and potentially an insulin sensitizer via activation of PPARγ and upregulation of glucose uptake.

Inhibiting geranylgeranylation increases neurite branching and differentially activates cofilin in cell bodies and growth cones.

Inhibitors of the mevalonate pathway, including the highly prescribed statins, reduce the production of cholesterol and isoprenoids such as geranylgeranyl pyrophosphates. The Rho family of small guanine triphosphatases (GTPases) requires isoprenylation, specifically geranylgeranylation, for activation. Because Rho GTPases are primary regulators of actin filament rearrangements required for process extension, neurite arborization, and synaptic plasticity, statins may affect cognition or recovery from nervous system injury. Here, we assessed how manipulating geranylgeranylation affects neurite initiation, elongation, and branching in neuroblastoma growth cones. Treatment with the statin, lovastatin (20 µM), decreased measures of neurite initiation by 17.0 to 19.0 % when a source of cholesterol was present and increased neurite branching by 4.03- to 9.54-fold (regardless of exogenous cholesterol). Neurite elongation was increased by treatment with lovastatin only in cholesterol-free culture conditions. Treatment with lovastatin decreased growth cone actin filament content by up to 24.3 %. In all cases, co-treatment with the prenylation precursor, geranylgeraniol (10 µM), reversed the effect of lovastatin. In a prior work, statin effects on outgrowth were linked to modulating the actin depolymerizing factor, cofilin. In our assays, treatment with lovastatin or geranylgeraniol decreased cofilin phosphorylation in whole cell lysates. However, lovastatin increased cofilin phosphorylation in cell bodies and decreased it in growth cones, indicating differential regulation in specific cell regions. Together, we interpret these data to suggest that protein geranylgeranylation likely regulates growth cone actin filament content and subsequent neurite outgrowth through mechanisms that also affect actin nucleation and polymerization.

Mevalonate deprivation mediates the impact of lovastatin on the differentiation of murine 3T3-F442A preadipocytes.

The statins competitively inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase activity and consequently the synthesis of mevalonate. The use of statins is associated with insulin resistance, presumably due to the impaired differentiation and diminished glucose utilization of adipocytes. We hypothesize that mevalonate is essential to adipocyte differentiation and adipogenic gene expression. Adipo-Red assay and Oil Red O staining showed that an eight-day incubation with 0-2.5 µmol/L lovastatin dose-dependently reduced the intracellular triglyceride content of murine 3T3-F442A adipocytes. Concomitantly, lovastatin downregulated the expression of peroxisome proliferator-activated receptor γ (Pparγ), leptin (Lep), fatty acid binding protein 4 (Fabp4), and adiponectin (AdipoQ) as measured by quantitative real-time polymerase chain reaction (real-time qPCR). The expression of sterol regulatory element binding protein 1 (Srebp-1), a transcriptional regulator of Pparγ and Lep genes, was also suppressed by lovastatin. Western-blot showed that lovastatin reduced the level of CCAAT/enhancer binding protein α (C/EBPα) while inducing a compensatory over-expression of HMG CoA reductase. The impact of lovastatin on intracellular triglyceride content and expression of the adipogenic genes was reversed by supplemental mevalonate. Mevalonate-derived metabolites have essential roles in promoting adipogenic gene expression and adipocyte differentiation.

Novel insights of dietary polyphenols and obesity.

The prevalence of obesity has steadily increased over the past three decades both in the United States and worldwide. Recent studies have shown the role of dietary polyphenols in the prevention of obesity and obesity-related chronic diseases. Here, we evaluated the impact of commonly consumed polyphenols, including green tea catechins, especially epigallocatechin gallates, resveratrol and curcumin, on obesity and obesity-related inflammation. Cellular studies demonstrated that these dietary polyphenols reduce viability of adipocytes and proliferation of preadipocytes, suppress adipocyte differentiation and triglyceride accumulation, stimulate lipolysis and fatty acid ß-oxidation, and reduce inflammation. Concomitantly, the polyphenols modulate signaling pathways including the adenosine-monophosphate-activated protein kinase, peroxisome proliferator activated receptor γ, CCAAT/enhancer binding protein α, peroxisome proliferator activator receptor gamma activator 1-alpha, sirtuin 1, sterol regulatory element binding protein-1c, uncoupling proteins 1 and 2, and nuclear factor-κB that regulate adipogenesis, antioxidant and anti-inflammatory responses. Animal studies strongly suggest that commonly consumed polyphenols described in this review have a pronounced effect on obesity as shown by lower body weight, fat mass and triglycerides through enhancing energy expenditure and fat utilization, and modulating glucose hemostasis. Limited human studies have been conducted in this area and are inconsistent about the antiobesity impact of dietary polyphenols probably due to the various study designs and lengths, variation among subjects (age, gender, ethnicity), chemical forms of the dietary polyphenols used and confounding factors such as other weight-reducing agents. Future randomized controlled trials are warranted to reconcile the discrepancies between preclinical efficacies and inconclusive clinic outcomes of these polyphenols.