Iron Supplementation Delays Aging And Extends Cellular Lifespan Through Potentiation Of Mitochondrial Function Ⅱ

3.2. Iron Supplementation Increases Oxidative Stress Resistance

Click Here To Get More Info About How Cistanche Reduce Oxidative stress  in your body

Cellular aging is associated with increased oxidative stress that damages the biological systems and mitigates age-related diseases [34]. Since iron supplementation delayed aging, we investigated its effect on oxidative stress. We used an oxidative stress inducer compound, hydrogen peroxide (H2O2), to test the oxidative resistance of cells. Cells were first grown in the presence of iron to the stationary phase stage (72 h) in SD medium. After that, cells were washed and incubated with different concentrations of H2O2 in the YPD medium and grew for 24 h. Oxidative stress resistance was analyzed by comparing the cell growth of the H2O2-treated cells with the non-treated control. We found that cells supplemented with iron were resistant to oxidative stress compared to the control (Figures 2a and S3). To confirm that iron supplementation provides resistance to oxidative stress, BPS was added to iron and cell growth with H2O2 was analyzed. The addition of BPS reduced the oxidative stress resistance of iron-supplemented cells (Figure 2b). These results correlate with the role of iron supplementation in delaying aging and extending the cellular lifespan.

3.3. Iron Supplementation Potentiates Mitochondrial Functions

Mitochondria are the major cellular hub for iron utilization and metabolism [35–38]. A decline in mitochondrial functions is associated with aging [39–43]. Iron serves as a cofactor of several mitochondrial proteins, including iron-sulfur clusters and heme-containing proteins [44–46]. These iron-containing proteins are involved in the mitochondrial tricarboxylic acid (TCA) cycle and electron transport chain (ETC) (Figure 3a). We investigated whether the increase in cellular lifespan by iron supplementation required mitochondrial functions. To test this, we analyzed the expression of mitochondrial TCA cycle genes. We found that iron supplementation signifificantly induces the expression of several TCA cycle genes (Figures 3b and S4a). TCA-cycle metabolites α-ketoglutarate and oxaloacetate can produce glutamate and aspartate by cataplerotic reactions in mitochondria (Figure 3a). These amino acids are utilized in the biosynthesis of proteins, lipids, and nucleotides [47–49]. Interestingly, the expression of glutamate (GDH1 and GDH3) and aspartate (AAT1 and AAT2) biosynthetic genes was decreased in iron-supplemented cells (Figure 3c). This expression profile suggests that iron supplementation promotes preservation instead of the consumption of TCA cycle intermediates. In agreement with this idea, gene expression of the mitochondrial anaplerotic pathway genes (PYC1, PYC2, and GDH2) was signifificantly increased in iron-supplemented cells (Figure 3c). PYC1 and PYC2 encode pyruvate carboxylase that converts pyruvate to oxaloacetate. GDH2 encodes glutamate dehydrogenase which synthesizes α-ketoglutarate from glutamate. Together, these results suggest that iron supplementation configures the cells in a metabolic state that favors anaplerosis and prevents cataplerosis to boost the TCA cycle metabolites.

Figure 3. Iron supplementation enhances mitochondrial functions. (a) An overview of the mitochondrial TCA (tricarboxylic acid) cycle and ETC (electron transport chain) with the major cataplerotic and anaplerotic reactions is illustrated. (b) The prototrophic yeast strain was incubated with 100 µM FeSO4 and grown in an SD medium for 8 h at 30 ◦C. RNA was extracted from the cultures and the expression of the indicated TCA cycle genes was analyzed by quantitative RT-PCR. (c) Expression analysis of cataplerotic genes (GDH1, GDH3, AAT1, and AAT2) and anaplerotic genes (PYC1, PYC2, and GDH2) was done by quantitative RT-PCR. (d) Expression analysis of ETC genes was done by quantitative RT-PCR. Statistical significance (* p < 0.05) was determined by Student’s t-test. 

TCA cycle reactions generate NADH and FADH2 which are oxidized by ETC complexes I and II and required for the functionality of the TCA cycle (Figure 3a). Although S. cerevisiae lacks complex I, reducing equivalents are transferred to the respiratory chain through NADH dehydrogenases. Succinate dehydrogenase plays a central role and participates in both the TCA cycle and the ETC complex II (Figure 3a). Strikingly, the expression of succinate dehydrogenase (SDH1 and SDH2) was highly upregulated among all analyzed TCA cycle genes (Figure 3b,d). These results indicate that the TCA cycle flux continues towards ETC instead of accumulating a particular intermediate. Likewise, the expression of all other genes of ETC complexes was highly upregulated in iron-supplemented cells (Figure 3d). ETC is associated with the generation of reactive oxygen species (ROS), which regulate the expression of the SOD2 gene [50]. It encodes a manganese-superoxide dismutase (MnSOD) which is the principal scavenger of mitochondrial superoxide. SOD2 gene expression was upregulated in iron-supplemented cells (Supplementary Figure S4a) which is consistent with the expression of ETC genes. We further analyzed the mitochondrial membrane potential (MMP) which is generated by the proton pumps of ETC complexes I, III, and IV. We found that iron supplementation increased the MMP of the cells (Supplementary Figure S4b). We then examined the structure of mitochondria using fluorescence microscopy. We found that iron supplementation prevents the fragmentation of mitochondria (Supplementary Figure S4c). Altogether, these results indicate that iron supplementation potentiates the mitochondrial functions of cells. 

3.4. Iron Supplementation Increases the ATP Level Required for Extension of Cellular Lifespan

Mitochondrial TCA cycle reactions produce reducing equivalents NADH and FADH2 which transfer electrons to ETC and generate adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS) (Figure 3a). Since the expression of the TCA cycle and ETC genes were enhanced by iron supplementation, we tested its effect on ATP synthesis. In agreement with the expression profile of the mitochondrial TCA cycle, ETC gene expression and ATP levels were high in iron-supplemented cells (Figure 4a). We then asked whether ATP is required for the extension of the lifespan of iron-supplemented cells. We inhibited the ATP synthesis and measured the CLS of yeast. Antimycin A (AMA) is an inhibitor of ATP synthesis which binds to complex III and blocks electron transfer in the mitochondrial ETC [51]. We first examined the ATP level and found that AMA treatment inhibits ATP synthesis (Figure 4b). We further tested the effect of AMA on the cellular lifespan of iron-supplemented cells. Yeast cells were incubated with iron and AMA in the SD medium. The growth of cells reached saturation after 24 h (Supplementary Figure S5). Subsequently, we measured the survival and found that AMA treatment inhibits the iron supplementation-mediated extension of lifespan (Figure 4c). Together, these findings reveal that iron supplementation increases the ATP level which is required for the extension of cellular lifespan. We also observed that the lifespan of AMA-treated cells was lower than the control (Figure 4c), further confirming that the inability to synthesize ATP compromised lifespan.

3.5. Iron Supplementation Prevents Accelerated Aging of AMPK Knockout Mutant

AMPK is the master regulator of cellular energy homeostasis [20,21]. The highly conserved human analog of AMPK in yeast S. cerevisiae is the Snf1 protein [52]. AMPK activates mitochondrial functions to produce ATP under energy-limited conditions. Recent reports have shown that the decline in mitochondrial functions with age occurs in part through the impaired activity of AMPK in different aged organisms [53,54]. Thus, the absence of AMPK activity affects mitochondrial functions and compromises numerous cellular functions including metabolism, resistance to stress, and cell survival which are the most critical determinants of aging and lifespan. Consistent with previous studies, we found that the snf1 knockout mutation compromised ATP level, resistance to oxidative stress, and lifespan (Figure 5a–c). 

Figure 5. Iron supplementation rescues the accelerated aging of the AMPK knockout mutant. The yeast prototrophic wild-type and AMPK knockout mutant (snf1∆) strains were grown to stationary phase in SD medium for 72 h at 30 ◦C. (a) ATP analysis of wild-type and snf1∆ strains. (b) Oxidative stress analysis of wild-type and snf1∆ strains with different H2O2 concentrations. (c) Chronological lifespan (CLS) analysis of wild-type and snf1∆ strains. (d) ATP analysis of wild-type and snf1∆ strains incubated with different concentrations of FeSO4. (e) Oxidative stress analysis of wild-type and snf1∆ strains incubated with different concentrations of FeSO4. (f) CLS analysis of wild-type and snf1∆ strains incubated with different concentrations of FeSO4. Statistical significance (* p < 0.05) was determined by Student’s t-test. 

Since the snf1 mutant is defective in mitochondrial functions, we tested whether iron supplementation can rescue the accelerated aging phenotypes. Therefore, we supplemented the iron to snf1 mutant and analyzed the ATP level, oxidative stress, and lifespan. We found that iron supplementation increased the ATP level, resistance to oxidative stress, and lifespan (Figure 5d–f). Altogether, these findings confirmed that iron supplementation delays aging and extends cellular lifespan by increasing mitochondria functions. 

4. Discussion

Nutrients determine the functional status of cells and the deficiency of essential nutrients compromises human health [55,56]. Furthermore, nutrients are the major regulators of cellular metabolism which controls several biological processes including aging, a major risk factor of several chronic diseases. A decline in metabolic activity is one of the hallmarks of aging [10]. Recent research in different organisms, including mammals, has demonstrated that delaying aging and increasing health span is feasible by anti-aging interventions including rapamycin and metformin drug administration [11,16]. These drugs target the nutrient-sensing complexes TORC1 and AMPK which are the important metabolic regulators of the cells [11,16]. 

Iron is an essential nutrient involved in several crucial metabolic reactions in the cells [23–26]. Iron deficiency impairs metabolic activity resulting in compromised cellular functions, leading to many diseases, including anemia, cognitive impairment, and loss of muscle strength [26,57–59]. Iron deficiency is widespread in elderly populations aged ≥65 years [60–62]. 

Since iron regulates metabolic processes, we investigated its role in aging. We utilized yeast as a model organism to examine the role of iron in chronological aging. We investigated the effect of iron supplementation on the CLS of yeast. We found that both FeSO4 and FeCl3 increased the cellular lifespan. Using different salts of sulfate, chloride, and iron chelator, we confirmed that the cellular lifespan is extended by iron. Aging is related to a gradual accumulation of oxidative stress, which is harmful to cellular functions and decreases cell survival [34]. Since we found that iron supplementation delayed aging, we tested whether it could provide resistance to oxidative stress. To examine the oxidative stress phenotype, we treated the cells with the oxidative inducer agent H2O2 and measured the cell survival. We found that iron supplementation increased oxidative stress resistance compared to control. These findings correlate with the role of iron supplementation in the extension of lifespan. 

We further unraveled the anti-aging mechanism of iron supplementation. Mitochondria are the main cellular consumers of iron utilization and metabolism [35–38]. We first analyzed the expression of mitochondrial TCA cycle genes. We found that the expression of almost all TCA cycle genes was upregulated by iron supplementation. Importantly, iron supplementation downregulated the expression of mitochondrial anaplerosis and upregulated cataplerotic metabolic genes. These results reveal that iron supplementation enhances the synthesis of TCA cycle intermediates and prevents their cellular utilization. These findings supported the anti-aging activity of iron supplementation, as regular export of TCA cycle intermediates affects mitochondrial integrity [47]. Moreover, replenishing the mitochondrial carbon pool is essential to maintain the mitochondrial functions required for survival during cellular aging.

The TCA cycle intermediate α-ketoglutarate has been shown to extend the lifespan of different organisms [63]. However, we found that iron supplementation increased the expression of α-ketoglutarate dehydrogenase (KGD1 and KGD2) which converts α- ketoglutarate to form succinyl-CoA. Moreover, we observed that the expression of succinate dehydrogenase genes (SDH1 and SDH2) was highly upregulated among other tested genes of the TCA cycle. Importantly, succinate dehydrogenase participates in both the TCA cycle and the ETC complex II. These results suggested that instead of accumulating a particular TCA cycle intermediate, the anti-aging activity of iron supplementation could involve the ETC pathway.

Since the TCA cycle is functionally connected with ETC, we analyzed the expression of ETC genes. We found that iron supplementation highly upregulated the expression of ETC genes. TCA cycle products NADH and FADH2 are oxidized by ETC complexes and generate ATP through OXPHOS. We found that iron supplementation increases the cellular ATP level, which is correlated with the upregulation of the TCA cycle and ETC genes. Next, we elucidated whether the increase in ATP level by iron supplementation is required for the extension of cellular lifespan. We found that lifespan extension by iron supplementation was abolished by inhibiting ATP synthesis. Thus, these fifindings suggest that iron supplementation increases the level of ATP which is required for the extension of cellular lifespan. Our results are consistent with the previous reports showing the role of ATP in the cellular lifespan [51,64,65]. Further, we utilized iron supplementation to enhance mitochondrial functions and rescued the short lifespan and oxidative stress-sensitive phenotype of the AMPK mutant. Altogether, these results revealed that iron supplementation potentiates the mitochondrial functions that delay aging and increase the lifespan of cells.

Recent studies have shown that iron supplementation restores the mitochondrial defect of lysosome-impaired mutants and prevents mitochondrial decline during aging [66,67]. Thus, our results support the previous findings that iron supplementation improves mitochondrial functions. Interestingly, one of the earlier studies showed that iron supplementation rescued the accelerated replicative aging of lysosome-impaired mutants; however, the effect on the wild-type cells was not included in the report [67]. Nevertheless, our results are correlated with previous findings, and despite different aging models (chronological aging), we found that iron supplementation delays aging and increases cellular lifespan. Thus, collectively, our results and previous reports clearly suggest that iron supplementation could be a potential therapeutic to target aging and increase healthspan. 

Supplementary Materials:  Figure S1: Growth and chronological lifespan analysis of iron-supplemented yeast cells; Figure S2: Growth analysis of different iron, sulfate, and chloride-containing salts-supplemented yeast cells; Figure S3: Oxidative stress resistance analysis of iron-supplemented yeast cells. Figure S4: Mitochondrial gene expression, membrane potential, and structure analysis of iron-supplemented yeast cells; Figure S5: Growth analysis of iron and antimycin A-supplemented yeast cells; Table S1: List of primers used for RT-PCR in this study.

Author Contributions: J.L.J.: Methodology, Formal analysis, Investigation, and Reviewing; T.C.Y.N.: Methodology, Formal analysis, Investigation, and Reviewing; F.N.: Validation, Formal analysis, and Reviewing; F.E.: Reviewing, Editing, and Supervision; M.A.: Conceptualization, Data curation, Methodology, Writing, Reviewing, Editing, and Supervision. All authors have read and agreed to the published version of the manuscript. 

Funding: This work was supported by Bioinformatics Institute (BII), A*STAR Career Development Fund (C210112008), and the Global Healthy Longevity Catalyst Awards grant (MOH-000758-00). 

Acknowledgments: We thank Maurer-Stroh Sebastian, Lee Hwee Kuan, Loo Lit Hsin, Chiam Keng Hwee, and Prakash Arumugam for backing this research. 

Conflicts of Interest: The authors declare no competing financial and non-financial interests.

References 

1. Bloom, D.E.; Canning, D.; Lubet, A. Global Population Aging: Facts, Challenges, Solutions & Perspectives. Chall. Solut. Perspect. Daedalus 2015, 144, 80–92. 

2. United Nations Department of Economic and Social Affairs, Population Division. World Population Prospects 2019; Highlights (ST/ESA/SERA/423); UN: New York, NY, USA, 2019.

3. Robine, J. Ageing Populations: We Are Living Longer Lives, but Are We Healthier? United Nations, Department of Economics and Social Affairs, Population Division: New York, NY, USA, 2021; UN DESA/POP/2021/TP/NO. 

4. Niccoli, T.; Partridge, L. Ageing as a risk factor for disease. Curr. Biol. 2012, 22, R741–R752. [CrossRef] [PubMed] 

5. Hou, Y.; Dan, X.; Babbar, M.; Wei, Y.; Hasselbalch, S.G.; Croteau, D.L.; Bohr, V.A. Ageing as a risk factor for neurodegenerative disease. Nat. Rev. Neurol. 2019, 15, 565–581. [CrossRef] [PubMed] 

6. Harman, D. The aging process: Major risk factor for disease and death. Proc. Natl. Acad. Sci. USA 1991, 88, 5360–5363. [CrossRef] [PubMed] 

7. Partridge, L.; Deelen, J.; Slagboom, P.E. Facing up to the global challenges of ageing. Nature 2018, 561, 45–56. [CrossRef] [PubMed] 8. Fontana, L.; Kennedy, 

B.K.; Longo, V.D.; Seals, D.; Melov, S. Medical research: Treat ageing. Nature 2014, 511, 405–407. [CrossRef] [PubMed] 

9. Fontana, L.; Partridge, L.; Longo, V.D. Extending healthy life span-from yeast to humans. Science 2010, 328, 321–326. [CrossRef] [PubMed] 

10. López-Otín, C.; Blasco, M.A.; Partridge, L.; Serrano, M.; Kroemer, G. The hallmarks of aging. Cell 2013, 153, 1194–1217. [CrossRef] 

11. Partridge, L.; Fuentealba, M.; Kennedy, B.K. The quest to slow aging through drug discovery. Nat. Rev. Drug Discov. 2020, 19, 513–532. [CrossRef]

12. Zimmermann, A.; Hofer, S.; Pendl, T.; Kainz, K.; Madeo, F.; Carmona-Gutierrez, D. Yeast as a tool to identify anti-aging compounds. FEMS Yeast Res. 2018, 18, foy020. [CrossRef] 

13. Kaeberlein, M.; Burtner, C.R.; Kennedy, B.K. Recent developments in yeast aging. PLoS Genet. 2007, 3, e84. [CrossRef] 

14. Longo, V.D.; Shadel, G.S.; Kaeberlein, M.; Kennedy, B. Replicative and chronological aging in Saccharomyces cerevisiae. Cell Metab. 2012, 16, 18–31. [CrossRef] 15. Longo, V.D.; Fabrizio, P. Chronological Aging in Saccharomyces cerevisiae. Aging Res. Yeast 2011, 57, 101–121. [CrossRef] 

16. Kulkarni, A.S.; Gubbi, S.; Barzilai, N. Benefifits of Metformin in Attenuating the Hallmarks of Aging. Cell Metab. 2020, 32, 15–30. [CrossRef] 

17. Alfatah, M.; Wong, J.H.; Krishnan, V.G.; Lee, Y.C.; Sin, Q.F.; Goh, C.J.H.; Kong, K.W.; Lee, W.T.; Lewis, J.; Hoon, S.; et al. TORC1 regulates the transcriptional response to glucose and developmental cycle via the Tap42-Sit4-Rrd1/2 pathway in Saccharomyces cerevisiae. BMC Biol. 2021, 19, 1–22. [CrossRef] 

18. Saxton, R.A.; Sabatini, D.M. mTOR signaling in growth, metabolism, and disease. Cell 2017, 168, 960–976. [CrossRef]