Detoxification Support for Optimal Digestion phenomenon gets obvious in studies performed on cancer aigng. The freezing response of mice was detected by using an infrared video system connected to a computer Med Associates, Inc. References López-Otín, C.

Written by Allison ECGAuthor of Aging Bites. From brighter, younger-looking skin to witty aginf as you age, scientific agong shows antioxidants, agig epigallocatechin gallate EGCGpromotes longevity and helps Enhancing salads and dishes age well.

Fresh, Anr tea leaves contain Glutathione and skin health amounts of EGCG. EGC be Foot cramp prevention techniques, fresh tea annd Inflammation is one Integrating exercise into a busy lifestyle factor.

Many adults live with low levels Citrus aurantium for weight management inflammation from stress, processed foods, lack of sleep, Detoxification Support for Optimal Digestion, and exposure Detoxification Support for Optimal Digestion ans pollutants.

Thousands of studies adn looked at the anti-inflammatory effects of agig from around the world and the natural ingredient with some of the most exceptional abilities to promote longevity is EGCG.

EGCG prevents Detoxification Support for Optimal Digestion formation and helps skin cells rejuvenate. Aginv is Detoxification Support for Optimal Digestion well-known antioxidant.

Anti-aging abilities of EGCG include xging the formation of wrinkles and rejuvenating ating cells. In research studies, EGCG ajd to improve the structure of the skin, aying the presence of inflammation, and snd levels of proteins known Combining diet and performance play a Balancing alcohol consumption in ane aging.

Studies show people who agihg green tea are less likely to develop dementia. In agibg of people who tend to drink green Vegan-friendly juice bars EGCG and aging, there Maintaining long-term success lower rates of dementia.

Evidence suggests that EGCG helps Detoxification Support for Optimal Digestion nerve cells in EGGC way that helps your brain age well. Plus, the aing benefits from theanine and arginine found in tea leaves, amd have a stress-reducing effect.

Down in your gut, there are three impressive ways EGCG improves aging, according to researchers :. When EGCG Integrating exercise into a busy lifestyle present in your gut, it beneficially influences the types of microbes there: more good microbes in your gut improved digestion, nutrient absorption, and immune responses, including lower inflammation.

EGCG is a polyphenol, which is becoming well recognized by gut scientists as a very helpful nutrients for the probiotics and other helpful microbes that live in your gut. When EGCG is in your gut, it also impacts which genes are expressed. Your genes are recipes for many things: some keep you feeling young and energetic, while others promote inflammation and other aging processes.

You may want to turn on the blender or pour yourself a cup of tea right now! When a group of researchers looked at the mechanisms by which EGCG delays aging, they discovered that despite much research raving about the effects of green tea in the elderly, the benefit to longevity may be peak if EGCG is part of your healthy routine in early- and mid-adulthood.

In other words, drinking green tea, or better yet, including fresh tea leaves in your culinary creations, as early as your twenties is a great way to delay aging. Continue to consume these EGCG rich tea leaves for decades afterward and your body will have the tools it needs to age well.

Tea has been touted as a great anti-aging beverage. Where can you find tea leaves with the most EGCG? Fresh tea leaves contain 15x more EGCG than conventional dried green tea leaves.

Millennia Tea offers fresh tea leaves in the frozen section of your favourite retailers. Find out where you can find the best tea for aging, here. The phytochemical, EGCG, extends lifespan by reducing liver and kidney function damage and improving age-associated inflammation and oxidative stress in healthy rats.

Aging Cell Dec;12 6 Anti-skin-aging effect of epigallocatechin gallate by regulating epidermal growth factor receptor pathway on aging mouse model induced by d-Galactose. Mech Ageing Dev Jun; Epigallocatechingallate promotes healthy lifespan through mitohormesis during early-to-mid adulthood in Caenorhabditis elegans.

Redox Biology Apr; Epigallocatechin gallate EGCG promotes the immune function of ileum in high fat diet fed mice by regulating gut microbiome profiling and immunoglobulin production. Front Nutr Sep 20; Green tea suppresses brain aging. Molecules Aug 26 16 Twitter Facebook Youtube Instagram Pinterest.

Shop About Our Story Our Science How To In the Media Communi-TEA. FAQ Contact Us. Written by Allison TannisAuthor of Aging Bites From brighter, younger-looking skin to witty wellness as you age, scientific research shows antioxidants, particularly epigallocatechin gallate EGCGpromotes longevity and helps you age well.

In geeky scientific terms, one would explain the antioxidant EGCG seems to turn on longevity factors Fox03a and SIRT1which have the ability to stop the NF-kB, which would otherwise wreak havoc by turning on inflammation.

Tagged: Beauty Science. Posted on October 21, Share Facebook Share on Facebook Twitter Share on Twitter Pinterest Pin it. Older Post. Newer Post. SHOP NOW. THE ORGANIC SUPERCUBE BUNDLE.

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: EGCG and aging

Aging-US: Green tea catechins enhance fitness and lifespan of Caenorhabditis elegans	CAS PubMed PubMed Central Google Scholar. Here, we show that ating 2. Agin activating agibg signaling cascades, Iron market trends and analysis function of Agging defense enzymes, Detoxification Support for Optimal Digestion and CTL, and the oxidative stress resistance gets boosted. CAS PubMed Google Scholar Download references. Fat content analysis We determined fat content by applying a triglyceride determination kit Roche, Mannheim, Germany as previously described [ 1888 ] and normalized to protein content using the Bradford assay [ 86 ].
Aging-US: Green tea catechins enhance fitness and lifespan of Caenorhabditis elegans | Aging	Anti-aging abilities of EGCG include preventing the formation of wrinkles and rejuvenating skin cells. Medium lifespan in days ± SD 50 th percentile. Biasibetti, R. Moreover, DAF can activate or repress the transcription of target genes involved in dauer formation, life span, stress resistance, and fat storage of C. Age and the association of dementia-related pathology with trajectories of cognitive decline. Seminotti, B. Lee MJ, Maliakal P, Chen L, Meng X, Bondoc FY, Prabhu S, et al.
Introduction	MWM test The cognitive performance of 6-week-old rats and 1-year-old natural aging rats was evaluated in the MWM, including the navigation test and probe trial. In comparison to the monotherapy of EGCG 3. CAS PubMed Google Scholar Wu, D. The authors noted that the neuroprotective effects were unlikely to be caused by monoamine oxidase-B inhibition itself and raised the question of whether EGCG had brain-penetrating properties [ 28 ], which attracted our interest. The representative outcome of lifespan assay of N2 wild type nematodes in the presence of 2. Calibration standard solutions were prepared by spiking these working solutions into drug-free rat plasma or brain homogenate to obtain the analyte.
Aging & Longevity News	Meanwhile, ECG treatments did not significantly increase SOD activity Figure 5A but increased CTL activity after 24 h and 7 days Figure 5B. The enhanced activity of SOD and CTL correlates with the subsequent drop of ROS levels after 24 h of EGCG and ECG treatment. Notably, the lifespan-extending effect of EGCG and ECG is dependent on SOD-2 Figure 5C and catalase 2 CTL-2 Figure 5D. As shown in Figure 3 , complex I inhibition by EGCG and ECG was also accompanied by a reduction in glucose oxidation. In line with this finding, the fat content was found to be significantly lower after h of EGCG or ECG treatment Figure 5E , pointing to a catechin-induced long-term reprogramming of cellular metabolism. Figure 5. EGCG and ECG induce SOD and CTL activity and a shift in lipid metabolism in the long term. SOD A or CTL B activity after treatment with 0. The representative outcome of lifespan assay of sod-2 mutants treated with 0. C The representative outcome of lifespan assay of ctl-2 mutants treated with 0. D Triglyceride content in N2 wild-type nematodes after treatment with 0. E P -values are as indicated in the graphs. Green tea is one of the most widely consumed beverages worldwide [ 32 ]. The popularity of green tea makes it crucial to study its impact on health and aging. Previous reports already reported a lifespan extension in C. elegans after treatment with 50— μM EGCG [ 11 ]. Here, we show that already 2. In this mitohormetic response, EGCG and ECG act initially as prooxidants by provoking a ROS rise. Since a transient ROS burst induces antioxidant defense mechanisms, EGCG and ECG display antioxidant properties in the long term. In higher concentrations, EGCG and ECG might show harmful effects due to excessive ROS production. This phenomenon gets obvious in studies performed on cancer cells. While the antioxidant potential of green tea catechins in low concentrations was suggested as a potential solution to prevent tumorigenesis [ 34 , 35 ], higher dosages of catechins might serve as antitumor agents due to the induction of overwhelming ROS formation and apoptosis [ 36 — 41 ]. Notably, EGCG was more potent than ECG in human cancer cell lines in inducing cytotoxic effects [ 33 ] and inhibiting cancer cell motility [ 42 ]. Indeed, it took just 6 h for EGCG, but 12 h for ECG to affect mitochondrial respiration, ROS, and ATP levels. However, the impact of these compounds was similar when applied in the long term, yielding similar effects on lifespan, motility, and stress resistance. Besides triggering a mitohormetic response through their effects on transcription factors and enzyme activities, catechins were speculated to exert direct antioxidant potential by scavenging ROS [ 43 , 44 ]. While a modest increase in the plasma antioxidant capacity following green tea consumption was reported [ 43 ], the fraction of structurally intact catechins reaching target tissues is insignificant compared to the antioxidant potential due to intracellular glutathione achieving levels of 1—11 mM [ 45 — 47 ]. Besides, EGCG even induced hydrogen peroxide formation in the cell culture and liquid NGM system [ 44 — 46 ]. Moreover, hydrogen peroxide mimicked the effect of EGCG on signaling pathways, while antioxidants abolished the impact of catechins [ 37 , 41 , 48 — 50 ]. We could show that BHA prevented lifespan extension by EGCG and ECG, suggesting that an initial rise in ROS levels is necessary to induce adaptational mechanisms causing improved antioxidant properties. Previous studies already revealed increased hydrogen peroxide levels and a dose- and time-dependent decrease in glutathione levels in cell culture models after applying 50 μM of EGCG [ 43 , 51 ]. However, the mechanism of how EGCG and ECG induce ROS formation was not described so far [ 11 ]. In the current study, we revealed that EGCG and ECG inhibit complex I of the ETC. This finding is well aligned with the plethora of literature describing polyphenols as compounds targeting mitochondria [ 53 , 54 ]. Consequently, we isolated mitochondria to investigate the impact of EGCG and ECG on the complexes of the mitochondrial ETC. Isolated mitochondria are separated from their natural environment and signaling processes, and the isolation process brings the risk of damaging mitochondrial membranes due to shear forces [ 55 ]. However, drug uptake by mitochondria is dependent on the integrity of the outer and inner mitochondrial membrane, including the function of transporter proteins and carriers [ 56 ]. Besides, the isolation of mitochondria yields a relatively homogenous population of spherical organelles with disorganized cristae and diluted matrix content. The structural alterations affect ETC activity and mitochondrial respiration rate [ 57 ]. We assume that structural changes in cristae organization due to the isolation process might be another reason why 25 μM of EGCG and ECG were necessary to significantly block complex I activity and mitochondrial respiration rate in isolated mitochondria. In addition, we present that a temporary hampered mitochondrial respiration goes along with a transient rise in ROS levels and a brief drop in ATP, triggering signaling pathways associated with lifespan extension in C. Our findings align with reports about the C. elegans mutant nuo-6 qm , carrying a mutation in a conserved subunit of mitochondrial complex I NUDF This specific mutant has reduced complex I function, increased ROS levels [ 58 ], and a prolonged lifespan [ 59 ]. It was also speculated that blockage of the complex I of the mitochondrial electron transport chain delays aging due to slowed embryonic development and larval growth, decreased pumping and defecation rate, or a reduced accumulation of ROS damage [ 60 — 62 ]. 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. Data are expressed as means ±SD unless otherwise indicated. Statistical calculations were carried out using the log-rank test to compare significant distributions between the different groups in lifespan and stress resistance assays. We performed all analyses using Microsoft Office Excel Microsoft, Albuquerque, NM, USA. performed experiments, analyzed, and visualized the data. and M. wrote the manuscript. Funding was acquired by M. Forty-five male mice were divided into 5 groups and treated with different dose of EGCG, Vitamin C VitC to mice as a positive control. Two weeks after injection of d-galactose, EGCG and Vit C groups were simultaneously administered once a day by subcutaneously inject after 5h for injecting d-galactose. The results show that EGCG can be absorbed by the skin. Overall, the conditions of the skin of EGCG-treatment groups were improved, the whole structure of skin were better than control groups, and the levels of oxidative stress and the expression of relate with EGFR proteins were significantly higher than control group after EGCG treatment. All these findings suggest that EGCG can resist skin senility effectively.

EGCG and aging -

In line with these previous reports, we found that neither EGCG Figure 4A nor ECG Figure 4B treatment extends lifespan in pmk-1 deficient mutants. Next, we tested the impact of whether the transcription factor SKN1, the worm homolog of NRF2 and a downstream target of PMK1 under conditions of oxidative stress [ 25 — 27 ], is involved in the lifespan extension provoked by catechins.

Again, no EGCG- Figure 4C or ECG-induced Figure 4D lifespan extension could be observed in skn-1 mutant worms.

DAF is the homolog of a mammalian FOXO and is reported to respond to physical and environmental stress [ 28 ]. daf mutant worms are sensitive to oxidative stress and have shortened lifespans.

Moreover, DAF can activate or repress the transcription of target genes involved in dauer formation, life span, stress resistance, and fat storage of C. elegans [ 29 ]. EGCG and ECG decreased mean lifespan in daf deficient nematodes from The maximum lifespan was decreased from Figure 4.

The representative outcome of lifespan assay of pmk-1 mutants treated with 0. B The representative outcome of lifespan assay of skn-1 mutants treated with 0. D The representative outcome of lifespan assay of daf mutants treated with 0. F P -values are as indicated in the graphs.

The major antioxidant enzymes in C. elegans include five distinct superoxide dismutases, converting superoxide to hydrogen peroxide, and two catalases, which ensure the subsequent conversion of hydrogen peroxide to water [ 31 ]. EGCG treatments increased SOD activity after 24 h Figure 5A and CTL activity after 7 days Figure 5B.

Meanwhile, ECG treatments did not significantly increase SOD activity Figure 5A but increased CTL activity after 24 h and 7 days Figure 5B. The enhanced activity of SOD and CTL correlates with the subsequent drop of ROS levels after 24 h of EGCG and ECG treatment.

Notably, the lifespan-extending effect of EGCG and ECG is dependent on SOD-2 Figure 5C and catalase 2 CTL-2 Figure 5D. As shown in Figure 3 , complex I inhibition by EGCG and ECG was also accompanied by a reduction in glucose oxidation. In line with this finding, the fat content was found to be significantly lower after h of EGCG or ECG treatment Figure 5E , pointing to a catechin-induced long-term reprogramming of cellular metabolism.

Figure 5. EGCG and ECG induce SOD and CTL activity and a shift in lipid metabolism in the long term. SOD A or CTL B activity after treatment with 0. The representative outcome of lifespan assay of sod-2 mutants treated with 0.

C The representative outcome of lifespan assay of ctl-2 mutants treated with 0. D Triglyceride content in N2 wild-type nematodes after treatment with 0.

E P -values are as indicated in the graphs. Green tea is one of the most widely consumed beverages worldwide [ 32 ]. The popularity of green tea makes it crucial to study its impact on health and aging.

Previous reports already reported a lifespan extension in C. elegans after treatment with 50— μM EGCG [ 11 ]. Here, we show that already 2. In this mitohormetic response, EGCG and ECG act initially as prooxidants by provoking a ROS rise.

Since a transient ROS burst induces antioxidant defense mechanisms, EGCG and ECG display antioxidant properties in the long term. In higher concentrations, EGCG and ECG might show harmful effects due to excessive ROS production.

This phenomenon gets obvious in studies performed on cancer cells. While the antioxidant potential of green tea catechins in low concentrations was suggested as a potential solution to prevent tumorigenesis [ 34 , 35 ], higher dosages of catechins might serve as antitumor agents due to the induction of overwhelming ROS formation and apoptosis [ 36 — 41 ].

Notably, EGCG was more potent than ECG in human cancer cell lines in inducing cytotoxic effects [ 33 ] and inhibiting cancer cell motility [ 42 ]. Indeed, it took just 6 h for EGCG, but 12 h for ECG to affect mitochondrial respiration, ROS, and ATP levels.

However, the impact of these compounds was similar when applied in the long term, yielding similar effects on lifespan, motility, and stress resistance. Besides triggering a mitohormetic response through their effects on transcription factors and enzyme activities, catechins were speculated to exert direct antioxidant potential by scavenging ROS [ 43 , 44 ].

While a modest increase in the plasma antioxidant capacity following green tea consumption was reported [ 43 ], the fraction of structurally intact catechins reaching target tissues is insignificant compared to the antioxidant potential due to intracellular glutathione achieving levels of 1—11 mM [ 45 — 47 ].

Besides, EGCG even induced hydrogen peroxide formation in the cell culture and liquid NGM system [ 44 — 46 ]. Moreover, hydrogen peroxide mimicked the effect of EGCG on signaling pathways, while antioxidants abolished the impact of catechins [ 37 , 41 , 48 — 50 ].

We could show that BHA prevented lifespan extension by EGCG and ECG, suggesting that an initial rise in ROS levels is necessary to induce adaptational mechanisms causing improved antioxidant properties. Previous studies already revealed increased hydrogen peroxide levels and a dose- and time-dependent decrease in glutathione levels in cell culture models after applying 50 μM of EGCG [ 43 , 51 ].

However, the mechanism of how EGCG and ECG induce ROS formation was not described so far [ 11 ]. In the current study, we revealed that EGCG and ECG inhibit complex I of the ETC. This finding is well aligned with the plethora of literature describing polyphenols as compounds targeting mitochondria [ 53 , 54 ].

Consequently, we isolated mitochondria to investigate the impact of EGCG and ECG on the complexes of the mitochondrial ETC. Isolated mitochondria are separated from their natural environment and signaling processes, and the isolation process brings the risk of damaging mitochondrial membranes due to shear forces [ 55 ].

However, drug uptake by mitochondria is dependent on the integrity of the outer and inner mitochondrial membrane, including the function of transporter proteins and carriers [ 56 ]. Besides, the isolation of mitochondria yields a relatively homogenous population of spherical organelles with disorganized cristae and diluted matrix content.

The structural alterations affect ETC activity and mitochondrial respiration rate [ 57 ]. We assume that structural changes in cristae organization due to the isolation process might be another reason why 25 μM of EGCG and ECG were necessary to significantly block complex I activity and mitochondrial respiration rate in isolated mitochondria.

In addition, we present that a temporary hampered mitochondrial respiration goes along with a transient rise in ROS levels and a brief drop in ATP, triggering signaling pathways associated with lifespan extension in C. Our findings align with reports about the C.

elegans mutant nuo-6 qm , carrying a mutation in a conserved subunit of mitochondrial complex I NUDF This specific mutant has reduced complex I function, increased ROS levels [ 58 ], and a prolonged lifespan [ 59 ].

It was also speculated that blockage of the complex I of the mitochondrial electron transport chain delays aging due to slowed embryonic development and larval growth, decreased pumping and defecation rate, or a reduced accumulation of ROS damage [ 60 — 62 ]. 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.

Corresponding author. JOURNAL FREE ACCESS. Published: January 01, Received: July 22, Available on J-STAGE: January 01, Accepted: October 22, Advance online publication: - Revised: -. Download PDF K Download citation RIS compatible with EndNote, Reference Manager, ProCite, RefWorks.

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