Modulation of Ca(2+) release and Ca(2+) oscillations in HeLa cells and fibroblasts by mitochondrial Ca(2+) uniporter stimulation.
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The recent availability of activators of the mitochondrial Ca(2+) uniporter allows direct testing of the influence of mitochondrial Ca(2+) uptake on the overall Ca(2+) homeostasis of the cell. We show here that activation of mitochondrial Ca(2+) uptake by 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) or kaempferol stimulates histamine-induced Ca(2+) release from the endoplasmic reticulum (ER) and that this effect is enhanced if the mitochondrial Na(+)-Ca(2+) exchanger is simultaneously inhibited with CGP37157. This suggests that both Ca(2+) uptake and release from mitochondria control the ability of local Ca(2+) microdomains to produce feedback inhibition of inositol 1,4,5-trisphosphate receptors (InsP(3)Rs). In addition, the ability of mitochondria to control Ca(2+) release from the ER allows them to modulate cytosolic Ca(2+) oscillations. In histamine stimulated HeLa cells and human fibroblasts, both PPT and kaempferol initially stimulated and later inhibited oscillations, although kaempferol usually induced a more prolonged period of stimulation. Both compounds were also able to induce the generation of Ca(2+) oscillations in previously silent fibroblasts. Our data suggest that cytosolic Ca(2+) oscillations are exquisitely sensitive to the rates of mitochondrial Ca(2+) uptake and release, which precisely control the size of the local Ca(2+) microdomains around InsP(3)Rs and thus the ability to produce feedback activation or inhibition of Ca(2+) release. (+info)
Identification of a flavonol 7-O-rhamnosyltransferase gene determining flavonoid pattern in Arabidopsis by transcriptome coexpression analysis and reverse genetics.
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Glycosylation plays a major role in the remarkable chemical diversity of flavonoids in plants including Arabidopsis thaliana. The wide diversity encoded by the large family-1 glycosyltransferase (UGT) gene family makes it difficult to determine the biochemical function of each gene solely from its primary sequence. Here we used transcriptome coexpression analysis combined with a reverse genetics approach to identify a gene that is prominent in determining the flavonoid composition of Arabidopsis. Using transcriptome coexpression analysis accessible on the ATTED-II public data base, the expression pattern of a UGT gene, UGT89C1, was found to be highly correlated with known flavonoid biosynthetic genes. No C-7 rhamnosylated flavonols were detected in either of two T-DNA ugt89c1 mutants. This specific metabolite deficiency in the mutants was complemented by stable transformation with the genomic fragment containing intact UGT89C1. Glutathione S-transferasefused recombinant UGT89C1 protein converted kaempferol 3-O-glucoside to kaempferol 3-O-glucoside-7-O-rhamnoside and recognized 3-O-glycosylated flavonols and UDP-rhamnose as substrates, but not flavonol aglycones, 3-O-glycosylated anthocyanins or other UDP-sugars. These results show that UGT89C1 is a flavonol 7-O-rhamnosyltransferase. The abundance of UGT89C1 transcripts in floral buds was consistent with the flavonoid accumulation of C-7 rhamnosylated flavonols in Arabidopsis organs. Our present study demonstrates that the integration of transcriptome coexpression analysis with a reverse genetic approach is a versatile tool for understanding a multigene family of a metabolic pathway in Arabidopsis. (+info)
The small polyphenolic molecule kaempferol increases cellular energy expenditure and thyroid hormone activation.
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Disturbances in energy homeostasis can result in obesity and other metabolic diseases. Here we report a metabolic pathway present in normal human skeletal muscle myoblasts that is activated by the small polyphenolic molecule kaempferol (KPF). Treatment with KPF leads to an approximately 30% increase in skeletal myocyte oxygen consumption. The mechanism involves a several-fold increase in cyclic AMP (cAMP) generation and protein kinase A activation, and the effect of KPF can be mimicked via treatment with dibutyryl cAMP. Microarray and real-time PCR studies identified a set of metabolically relevant genes influenced by KPF including peroxisome proliferator-activated receptor gamma coactivator-1alpha, carnitine palmitoyl transferase-1, mitochondrial transcription factor 1, citrate synthase, and uncoupling protein-3, although KPF itself is not a direct mitochondrial uncoupler. The cAMP-responsive gene for type 2 iodothyronine deiodinase (D2), an intracellular enzyme that activates thyroid hormone (T3) for the nucleus, is approximately threefold upregulated by KPF; furthermore, the activity half-life for D2 is dramatically and selectively increased as well. The net effect is an approximately 10-fold stimulation of D2 activity as measured in cell sonicates, with a concurrent increase of approximately 2.6-fold in the rate of T3 production, which persists even 24 h after KPF has been removed from the system. The effects of KPF on D2 are independent of sirtuin activation and only weakly reproduced by other small polyphenolic molecules such as quercetin and fisetin. These data document a novel mechanism by which a xenobiotic-activated pathway can regulate metabolically important genes as well as thyroid hormone activation and thus may influence metabolic control in humans. (+info)
Evaluation of a method to determine flavonol aglycones in Ginkgo biloba dietary supplement crude materials and finished products by high-performance liquid chromatography: collaborative study.
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An interlaboratory study was conducted for evaluation of a method to determine the flavonol aglycones quercetin, kaempferol, and isorhamnetin in Ginkgo biloba products. The method calculates total glycosides based on these aglycones formed after acid hydrolysis. Twelve matrixes were chosen for study by 12 collaborating laboratories in 2 countries. Test materials included crude leaf material, standardized dry powder extract, single and multiple entity finished products, ethanol and glycerol tinctures, and National Institute of Standards and Technology (NIST) standard reference materials (SRMs). Results from 11 laboratories were used for the final calculations. Eight of the 12 matrixes evaluated produced acceptable results for total flavonol glycosides, with HorRat scores ranging from 1.31 to 2.05; repeatability relative standard deviations (RSDr) from 1.46 to 4.14; and reproducibility relative standard deviations (RSDR) from 4.67 to 9.69. These 8 matrixes consisted primarily of simple dosage forms (e.g., dry powder extracts, crude leaf samples, liquid extracts, and SRMs) and a single tablet product (Ginkgo Awareness). Four additional matrixes, consisting of 3 tablets and 1 soft gel product (Ginkgold, Ginkoba, Ginkogen, and Ginkgo Phytosome, respectively), showed greater total flavonol glycoside HorRat scores in comparison, ranging from 2.39 to 5.13, with RSDr values from 2.83 to 8.16, and RSDR values from 8.53 to 20.4. Based on the results presented here, the method is recommended for Official First Action for determination of total flavonol glycosides calculated from quercetin, kaempferol, and isorhamnetin in dry powder extracts, crude leaf material, liquid extracts, and a select finished product, Ginkgo Awareness. (+info)
Dietary intakes of flavonols and flavones and coronary heart disease in US women.
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Dietary flavonols and flavones are subgroups of flavonoids that have been suggested to decrease the risk of coronary heart disease (CHD). The authors prospectively evaluated intakes of flavonols and flavones in relation to risk of nonfatal myocardial infarction and fatal CHD in the Nurses' Health Study. They assessed dietary information from the study's 1990, 1994, and 1998 food frequency questionnaires and computed cumulative average intakes of flavonols and flavones. Cox proportional hazards regression with time-varying variables was used for analysis. During 12 years of follow-up (1990-2002), the authors documented 938 nonfatal myocardial infarctions and 324 CHD deaths among 66,360 women. They observed no association between flavonol or flavone intake and risk of nonfatal myocardial infarction or fatal CHD. However, a weak risk reduction for CHD death was found among women with a higher intake of kaempferol, an individual flavonol found primarily in broccoli and tea. Women in the highest quintile of kaempferol intake relative to those in the lowest had a multivariate relative risk of 0.66 (95% confidence interval: 0.48, 0.93; p for trend = 0.04). The lower risk associated with kaempferol intake was probably attributable to broccoli consumption. These prospective data do not support an inverse association between flavonol or flavone intake and CHD risk. (+info)
Esterase inhibition by grapefruit juice flavonoids leading to a new drug interaction.
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Our previous studies described a newly identified potential of grapefruit juice (GFJ) in mediating pharmacokinetic drug interactions due to its capability of esterase inhibition. The current study identifies the active components in GFJ responsible for its esterase-inhibitory effect. The esterase-inhibitory potential of 10 constitutive flavonoids and furanocoumarins toward p-nitrophenylacetate (PNPA) hydrolysis was investigated. The furanocoumarins bergamottin, 6',7'-dihydroxybergamottin, and bergapten, and the glycoside flavonoids naringin and hesperidin, at concentrations found in GFJ or higher, did not inhibit the hydrolysis of PNPA by purified porcine esterase and human liver microsomes. However, the flavonoid aglycones morin, galangin, kaempferol, quercetin, and naringenin showed appreciable inhibition of PNPA hydrolysis in purified porcine esterase, and human and rat liver systems. In Caco-2 cells, demonstrated to contain minimal CYP3A activity, the permeability coefficient of the prodrugs lovastatin and enalapril was increased in the presence of the active flavonoids kaempferol and naringenin, consistent with inhibition of esterase activity. In rats, oral coadministration of kaempferol and naringenin with these prodrugs led to significant increases in plasma exposure to the active acids. In addition, in portal vein-cannulated rats, coadministration of lovastatin with kaempferol (10 mg/kg) led to a 154% and a 113% increase in the portal plasma exposure to the prodrug and active acid, respectively, compared with coadministration with water. The contribution of CYP3A inhibition was demonstrated to be minimal. Overall, a series of flavonoids present in GFJ are identified as esterase inhibitors, of which kaempferol and naringenin are shown to mediate pharmacokinetic drug interaction with the prodrugs lovastatin and enalapril due to their capability of esterase inhibition. (+info)
Dietary flavonoids induce MLL translocations in primary human CD34+ cells.
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Genetic abnormalities leading to infant leukemias already occur during fetal development and often involve rearrangements of the mixed-lineage leukemia (MLL) gene. These rearrangements resemble the aberrations observed in therapy-related leukemias following treatment with topoisomerase II (topoII)-inhibiting agents such as etoposide. Since flavonoids are potent topoII inhibitors, we examined the role of three widely consumed dietary flavonoids (quercetin, genistein and kaempferol) on the development of MLL rearrangements in primary human CD34(+) cells. Using the neutral Comet assay, we demonstrated a dose-dependent double-strand break (DSB) formation after exposure to flavonoids. An incorrect repair of these DSBs resulted in chromosomal translocations that co-localized with those identified in infant leukemias. Most of these translocations were formed by microhomology-mediated end joining. Moreover, in all but one translocation, SINE/Alu or LINE/L1 repetitive elements were present in at least one side of the breakpoint junction. Beside MLL translocations, fluorescence in situ hybridization analysis demonstrated monosomy or trisomy of MLL in 8-10% of the quercetin-exposed CD34(+) cells. Our study demonstrates that biologically relevant concentrations of flavonoids can induce MLL abnormalities in primary hematopoietic progenitor cells. This is particularly alarming knowing that the differences in metabolism and excretion rate between mother and fetus can lead to a higher flavonoid concentration on the fetal side. Therefore, it is important to raise public awareness and set guidelines for marketing flavonoid supplements to reduce the risk of infant leukemias. (+info)
New flavonoid glycosides from the leaves of Solidago altissima.
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Two new flavonoid glycosides kaempferol 3-O-beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside (1), and quercetin 3-O-beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside (2), together with six known flavonoid glycosides were isolated from the leaves of Solidago altissima L. grown in Kochi of Japan. The structure elucidation of the isolated compounds was performed by acid hydrolysis and spectroscopic methods including UV, IR, ESI-MS, 1D- and 2D-NMR experiments. (+info)