Anti-aging catechins -
At this stage, mitochondria are already undergoing a period of dramatic proliferation and massive mitochondrial DNA expansion [ 63 ]. Moreover, inhibiting respiratory chain components during adulthood did not provoke lifespan extension anymore [ 64 — 66 ].
Consequently, one has to assume that a temporary sub-lethal rise in mitochondrial ROS during early adulthood induces lifespan extension by provoking changes in the homeostasis of proteins [ 59 , 67 ] and metabolism [ 58 ].
Notably, glucose restriction by 2-deoxy-D-glucose 2-DG -mediated inhibition of glycolysis increases the lifespan in C. elegans in a ROS-dependent manner [ 18 ], suggesting that the temporary drop in ATP levels due to complex I inhibition is an additional trigger to prolong lifespan.
By activating these signaling cascades, the function of ROS defense enzymes, SOD and CTL, and the oxidative stress resistance gets boosted. Ahead of this report, SOD-3, DAF, and SKN-1 were already suggested as targets of EGCG due to enhanced expression [ 68 ] or translocation into the nucleus after respective compound treatment [ 48 ].
Notably, SKN-1 activation in neurons is necessary for dietary restriction-mediated lifespan extension [ 69 ]. DAF, the orthologue of mammalian FOXO, is a crucial regulator of longevity, metabolism, and dauer diapauses in C.
Consequently, it seems reasonable that the ROS-sensing p38 MAPK and the energy-sensing AMPK activate the respective signaling cascades after blockage of complex I by EGCG and ECG. Reports showed that AMPK activates p38 MAPK [ 73 ]. The long-term effects also included reduced fat content in C.
elegans after 5 days of catechin treatment. Align with this finding, inhibition of complex I and complex IV by rotenone and NaN3 reduced lipid accumulation in 3T3-L1 cells [ 74 ].
Moreover, a previous report revealed reduced body fat content in C. elegans after catechin treatment [ 75 ]. Besides, green tea catechins were associated with reduced obesity in zebrafish [ 76 ], mice [ 77 ], rats [ 78 , 79 ], and humans [ 80 , 81 ], suggesting a catechin-induced metabolic remodeling.
Clinical trials have already confirmed the safety of EGCG [ 7 ] and highlighted the potential in counteracting age-related cardiovascular and metabolic diseases [ 1 — 4 ].
Experiments in rodents studying physical and clinical parameters over time and further clinical trials are required to identify the best timing and dosage for administering catechins. Finally, these studies might characterize additional effects and downstream mechanisms of complex I inhibition.
Despite the promising results obtained in animal experiments, the low bioavailability of EGCG [ 7 ] still raises the question of whether green tea catechins can reliably provoke beneficial effects in humans.
Consequently, additional efforts might be needed to identify complex I inhibitors with increased bioavailability. We conclude that applying the green tea catechins EGCG and ECG at a low dose extends the lifespan of C.
elegans via inducing a mitohormetic response. In the long term, the re-wiring of these energy- and ROS-dependent pathways reduces the fat content and extends health- and lifespan. Figure 6. Green tea catechins enhance fitness and lifespan of Caenorhabditis elegance by complex I inhibition.
elegans strains used in the current study were obtained from the Caenorhabditis Genetics Center CGC, University of Minnesota. Nematodes were grown and maintained at 20°C in 10 cm Petri dishes on nematode growth media NGM , with Escherichia coli E.
coli OP50 bacteria as the food resource as previously described [ 18 , 82 , 83 ]. The strains used in this study included the following: N2 wild type , GA aak-2 ok , VC sir EGCG, ECG, and BHA dissolved in DMSO, reaching a stock concentration of 2. The NGM agar solution was autoclaved and subsequently cooled to 55°C, before supplements and compounds EGCG, ECG, BHA, or DMSO were added under continuous stirring.
The final concentration for compounds was calculated regarding the volume of agar, and the same volume of DMSO was added to control plates.
Agar plates were poured and dried, sealed with parafilm, and stored at 4°C. Before experiments, NGM plates were spotted with a bacterial lawn of heat-inactivated bacteria OP50 HIT to avoid interference by a potential xenobiotic-metabolizing activity of E.
To exclude any effects on development, the incubation period with compounds started at the L4 stage by transferring nematodes to the respective NGM plates [ 84 ]. Louis, MO, USA to prevent progeny formation. After 16 h, we transferred animals to respective treatment groups and harvested them at the indicated time points [ 18 ].
According to standard protocols, all lifespan assays were performed at 20°C as previously described [ 18 , 19 ]. Briefly, the C. Eggs of nematodes were transferred to NGM plates with fresh OP50 bacteria to allow hatching and development.
After approximately 64 h, at the L4 stage, we moved nematodes manually to freshly prepared NGM plates containing the respective compounds and supplied them with a lawn of OP50 HIT. During the first 10—14 days, nematodes were transferred to freshly prepared NGM treatment plates every day and later every second day.
Nematodes without any reaction to gentle stimulation were classified as dead. Nematodes that crawled off the plate or suffered from non-natural death like internal hatching were censored and excluded from statistics on the day of premature death.
Notably, for lifespan analysis using BHA, nematodes were propagated on BHA-containing NGM plates for four generations before synchronization; the same applied for the respective DMSO controls. Following the L4 stage, nematodes were treated with 0. Afterward, we transferred single worms into S-buffer containing 0.
Movements of single worms within the liquid system were recorded for 20 seconds by a digital CCD camera Moticam , Motic, St. Ingbert, Germany coupled microscope SMZ , Motic, St. Ingbert, Germany equipped with Motic Images Plus 2.
We analyzed the videos using the DanioTrack software Loligo Systems, Tjele, Denmark , subtracting the background and determining the center of gravity of all object pixels compared to the background.
Resistance to lethal oxidative stress by paraquat Sigma-Aldrich, Munich, Germany was assessed as previously described [ 18 , 19 ]. Briefly, worms were treated with 0. Afterward, we transferred worms into well plates: 6 nematodes in μl of S-buffer, containing freshly dissolved 50 mM paraquat.
Dead worms were scored every hour until all control worms were dead. Briefly, we treated worms with 0. Worms were also washed twice with S-buffer and transferred into the DW1 chamber to monitor oxygen consumption for 10 mins.
Afterward, we collected worms for Bradford protein determination [ 86 ]. Before the ROS measurement, MitoTracker Red CM-H2X ROS Invitrogen, Carlsbad, CA, USA incubation plates were prepared as previously described [ 19 ].
Worms were additionally washed twice with S-buffer and transferred to freshly prepared MitoTracker Red CM-H2X incubation NGM plates containing μl of OP50 HIT mixed with μl freshly prepared MitoTracker Red CM-H2X stock solution μM.
After 2 h at 20°C, worms were washed off MitoTracker Red CM-H2X incubation NGM plates and transferred to NGM agar plates with 0. Fluorescence intensity was measured on a microplate reader FLUOstar Optima, BMG Labtech, Offenburg, Germany using well-scanning mode ex: nm; em: nm.
We collected worms from plates for Bradford protein determination [ 86 ]. We placed an equal number of nematodes on the NGM plates containing 0. After collection and two subsequent washes in S-buffer, worm pellets were resuspended in the incubation buffer.
The latter were placed in 10 cm Petri dishes together with a second 4 cm Petri dish containing μl of 0. Hence, each 10 cm dish was equipped with two 4 cm dishes, one carrying nematodes and the other containing KOH.
We added nonradioactive glucose into each sample to reach a final concentration of 0. The 10 cm Petri dishes were covered, sealed with parafilm in an air-tight manner, and incubated at 20°C for 3 h. Subsequently, an aliquot of μl of KOH was immersed in 4.
to quantify the amount of trapped 14 CO 2. We treated nematodes with 0. After collection and washing with S-buffer twice, worm pellets were shock frozen in liquid nitrogen and grinded in a nitrogen-chilled mortar. The grinded samples were boiled with 4 M Guanidine-HCl at 99°C for 15 min to destroy ATPase activity [ 58 , 89 ].
ATP values were normalized to protein content using the Bradford assay [ 86 ]. After treating nematodes with 0. The produced formaldehyde was determined spectrophotometrically with 4-aminohydrazinomercapto-1, 2, 4-triazole Purpald, Applichem, Darmstadt, Germany.
We measured SOD activity photometrically with a tetrazolium salt, forming a water-soluble formazan dye upon reduction with a superoxide anion. We determined fat content by applying a triglyceride determination kit Roche, Mannheim, Germany as previously described [ 18 , 88 ] and normalized to protein content using the Bradford assay [ 86 ].
Briefly, worms were incubated with 0. We centrifuged μl of the homogenized extract and extracted the supernatant for protein determination. The heating was repeated once to dissolve all triglycerides. We measured the activity of complex I spectrophotometrically at nm in 1 ml of 25 mM potassium phosphate buffer containing 3.
Decylubiquinone and antimycin A were dissolved in DMSO as DCIP and NADH were dissolved in water as 10 mM for both. After being thawed, 30 μl of mitochondria were treated with μl of 10 mM Tris-Cl, pH 7.
Subsequently, 20 μl mitochondria fragments were preincubated in a μl incubation mixture without NADH for 3 mins. After 3 mins, we added 20 μl of 10 mM NADH into the incubation mixture and measured the absorbance at 20 s intervals for 2 mins.
Briefly, rodents were fasted overnight and killed by cervical dislocation. The washed liver fragments were placed into the tube with around 25 ml isolation buffer.
The loose-fitting pestle was inserted, pressed down, and lifted four times, and then the tight-fitting pestle was applied in the same way twice. The mixture was poured into the 50 ml polypropylene falcon tube and centrifuged at g for 10 min at 4°C. We carefully removed the fat on the top of the supernatant by using tissue paper.
The supernatant was extracted to a second polypropylene falcon tube centrifuged at g for 10 min at 4°C. Afterward, the fat was removed, the supernatant discarded, and the mitochondrial pellet resuspended in the remaining buffer. The suspension containing mitochondria was centrifuged again at g for 10 min at 4°C.
The supernatant was removed entirely, and the mitochondrial pellet was resuspended in μl isolation buffer as described above. The concentration of isolated mitochondria was determined with Bradford After recording basal respiration for 2 min, 0.
After ADP was wholly consumed, the oxygen consumption rate slowed down, 5 mM succinate, and ADP were added to study complex II, III, IV activity.
At the end of each measurement 60 nM FCCP were supplied to check the viability of mitochondria. Rapamycin Redux by Josh Mitteldorf. Dr Randy Smith, Antiaging Atlanta.
Aging, COVID and more: Interview with Dr. Elizabeth Blackburn , a member of the Editorial Board of Aging, won the Nobel Prize in Physiology or Medicine , while being a member of the board.
Elizabeth Blackburn co-authored a paper published in the first inaugural issue of Aging. Andrew V. Schally , Nobel Prize Laureate, published his paper in Aging. Shinya Yamanaka won the Nobel Prize in Physiology and Medicine Shinya Yamanaka co-authored a paper published in Aging.
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by contributing institutions or for the use of any information through the EurekAlert system. Journal Aging-US. DOI Article Title "Green tea catechins EGCG and ECG enhance the fitness and lifespan of Caenorhabditis elegans by complex I inhibition". JOURNAL FREE ACCESS. Published: May 25, Received: February 03, Available on J-STAGE: May 14, Accepted: February 15, Advance online publication: - Revised: -.
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