Iron Supplementation Delays Aging and Extends Cellular Lifespan through Potentiation of Mitochondrial Function

The prototrophic Saccharomyces cerevisiae CEN.PK wild-type strain was used in all experiments to avoid the effects of amino acid auxotrophy on cell growth and survival [27,28]. The gene knockout strain was generated by PCR-mediated homologous recombination whereby the entire locus was replaced by an amplified selection marker containing the upstream and downstream flanking sequences of the target gene [29]. PCR confirmed stable integration of the amplified selection marker.

The rich medium YPD contained 1% yeast extract, 2% peptone, and 2% glucose, YPD agar (2.5% Bacto agar), and the SD medium contained 6.7 g/L yeast nitrogen base with ammonium sulfate without amino acids (Difco) and 2% glucose. FeSO₄.7H₂O, FeCl₃, CaSO₄.2H₂O_, MgSO₄.7H₂O_, CaCl₂.2H₂O_, MgCl₂.6H₂O, and BPS were purchased from Sigma, and their stock solution was prepared in water. Antimycin A (Sigma, St. Louis, MO, USA), DiOC6(3) (3,3′-Dihexyloxacarbocyanine Iodide) (Invitrogen™, Waltham, MA, USA), and MitoTracker™ Deep Red stock (Invitrogen™) solution was prepared in dimethyl sulfoxide (Sigma). The final concentration of DMSO did not exceed 1% in any assay.

The wild-type and deletion strains were recovered from frozen glycerol stock on YPD agar medium at 30 °C. Yeast cells were grown in SD medium overnight at 30 °C with shaking at 220 rpm. The cell cultures grown overnight were diluted to OD600nm~0.2 in fresh SD medium to initiate growth assay in a 96-well plate or flask with or without chemicals and incubated at 30 °C. The cell growth (OD600nm) was measured at different time points using a microplate reader or spectrophotometer.

Chronological aging was determined by measuring the chronological lifespan (CLS) of yeast cells, as previously reported with slight modifications [30,31]. The CLS experiment was carried out on a 96-well plate. The overnight cell culture was diluted to OD600nm~0.2 in fresh SD medium and transferred into the 96-well plate containing different concentrations of chemicals and incubated at 30 °C. Cells were grown to a stationary phase which was considered Day1 (100% cell survival) for the CLS analysis. Yeast cells' survival was quantified at various age time points by an outgrowth assay. Chronologically aged cells (3 μL) at different age time points were transferred to a second 96-well plate. YPD medium (200 μL) was added to a 96-well plate and incubated for 24 h at 30 °C. Cell outgrowth was measured by absorbance (OD600nm) using the microplate reader. Cell survival for different age points was quantified relative to Day1 (considered 100% cell survival) using the calculation formula:Survival % = [Outgrowth OD600nm (age point)/Outgrowth OD600nm (Day1)] × 100

Yeast cells were grown to stationary phase stage (72 h) in SD medium. After that, cells were washed and diluted to OD600nm~0.2 in a YPD medium with different concentrations of H₂O₂ and grown at 30 °C. Oxidative stress resistance was analyzed by comparing the cell growth of H₂O₂ treated cells with the non-treated control.

Total RNA was extracted from cells using the RNeasy mini kit (Qiagen, Hilden, Germany) following the manufacturer's mechanical disruption protocol. RNA concentration and quality were determined by spectrophotometer (NanoDrop 2000 Thermo Scientific, Waltham, MA, USA). Typically, 1 μg total RNA was reverse transcribed into cDNA using QuantiTect Reverse Transcription Kit (Qiagen). During the synthesis of cDNA, two negative controls including without reverse transcriptase and RNA template were also performed. All RT-PCR was performed in a final volume of 20 μL containing 20 ng of cDNA using SYBR Fast Universal qPCR Kit (Kapa Biosystems, Boston, MA, USA) and analyzed using the Quant Studio 6 Flex system (Applied Biosystems, Waltham, MA, USA). The RT-PCR condition was one hold at {95 °C, 180 s}, followed by 40 cycles of {95 °C, 1 s}, and {60 °C, 20 s} steps. After amplification, a melting curve was performed to verify PCR specificity and the absence of primer dimers. The quantitative abundance of each gene was determined relative to the house-keeping transcript ACT1. The relative gene expression between the control and treated conditions was calculated using the 2^−ΔΔCt method [32]. A list of primers used for RT-PCR is provided in Supplementary Table S1.

Yeast cultures were washed with 1× PBS (phosphate buffered saline) and incubated with DiOC6(3) (100 nM) for 30 min in the dark. After incubation, cells were washed and resuspended in 1× PBS. The fluorescence reading (excitation at 482 nm, emission at 504 nm) of samples were measured by the microplate reader. The fluorescence intensity of each sample was normalized with OD600nm. For analysis of mitochondrial structure cells were washed with 1× PBS and incubated with MitoTracker deep red (100 nM) for 30 min in the dark. The samples were visualized with a fluorescence microscope at 100× magnification and the images were processed by ImageJ software.

Yeast cultures were mixed with tricholoroacetic acid to a final concentration of 5% and kept on ice for at least 5 min. Next, the cells were spun down, resuspended in 150 μL 5% trichloroacetic acid, and lysed with glass beads on Precellys^® 24 homogenizer (Bertin Technologies, Montiny Le Bretonne, France). ATP level was measured using an Enliten luciferin/luciferase reagent kit (Promega, Madison, WI, USA) in a luminometer (Biotek, Winusky, VT, USA). Protein concentration was estimated with Bradford reagent (Bio-rad, Hercules, CA, USA). The ATP level in the samples was normalized to total protein concentration.

All experiments were performed in three independent biological triplicates. Statistical analysis such as mean value, standard deviations, and statistical significance was calculated using GraphPad Prism 9 software.

We investigated the effect of Iron(II) sulfate (FeSO₄) supplementation on the chronological lifespan (CLS) of yeast in a 96-well plate. First, cells were incubated with different concentrations of FeSO₄ in a synthetic defined (SD) medium, and growth was analyzed at different time points (16 h, 24 h, and 48 h). Cell growth reached saturation approximately 24 h after incubating with FeSO₄ concentrations (Supplementary Figure S1a,b). Next, we determined the CLS of cells incubated with different concentrations of FeSO₄. We considered the 48 h stationary phase cell culture as Day1 for CLS analysis. The viability of aged cells at different points was normalized with Day1 (100% viable) and plotted on the survival graph. We found that different concentrations of FeSO₄ supplementation in the medium extended the CLS of yeast (Figure 1a and Figure S1c). We also tested Iron(III) chloride (FeCl₃) and found a similar result to FeSO₄ (Figure 1b and Figure S2a).

To clarify whether FeSO₄ and FeCl₃ salts, their components Iron(II) and Iron(III), or sulfate and chloride extend CLS, we examined the other sulfate and chloride-containing salts. Yeast cells were incubated with CaSO_4, MgSO_4, CaCl_2, MgCl₂ including FeSO_4, and FeCl₃ in the SD medium. Growth of cells incubated with different salts reached saturation after 24 h (Supplementary Figure S2b). We next measured the survival and found that except FeSO₄ and FeCl₃, other sulfate or chloride-containing salts did not extend the lifespan of yeast (Figure 1c). These results revealed that iron, but not sulfate and chloride, extend the CLS of yeast.

For further validation, we depleted the iron and analyzed the CLS of yeast. Bathophenanthrolinedisulfonic acid (BPS) is a specific iron chelator compound that sequesters the iron and leads to iron deficiency in the cells [33]. Yeast cells were incubated with iron and different concentrations of BPS in the SD medium. Cell growth of different concentrations reached saturation after 24 h (Supplementary Figure S2c). We further measured the cell survival and found that BPS addition reduced the lifespan of iron-supplemented cells (Figure 1d). Altogether, these results confirmed that iron supplementation increases the lifespan of yeast.

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 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 control (Figure 2a and Figure 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.

Mitochondria are the major cellular hub for iron utilization and metabolism [35,36,37,38]. A decline in mitochondrial functions is associated with aging [39,40,41,42,43]. Iron serves as a cofactor of several mitochondrial proteins, including iron-sulfur clusters and heme-containing proteins [44,45,46]. 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 significantly induces the expression of several TCA cycle genes (Figure 3b and Figure 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 [47,48,49]. 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 significantly 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.

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.

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. 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.

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 the 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).

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.