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Article

Isocitrate Dehydrogenase Alpha-1 Modulates Lifespan and Oxidative Stress Tolerance in Caenorhabditis elegans

by
Zhi-Han Lin
1,2,3,4,†,
Shun-Ya Chang
1,†,
Wen-Chi Shen
1,‡,
Yen-Hung Lin
1,‡,
Chiu-Lun Shen
5,6,7,
Sin-Bo Liao
1,
Yu-Chun Liu
1,
Chang-Shi Chen
8,
Tsui-Ting Ching
9 and
Horng-Dar Wang
1,5,10,*
1
Institute of Biotechnology, National Tsing Hua University, Hsinchu 300044, Taiwan
2
Institute of Chemistry, Academia Sinica, Taipei 115201, Taiwan
3
Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, Taipei 115201, Taiwan
4
Institute of Biochemical Sciences, National Taiwan University, Taipei 106319, Taiwan
5
Department of Life Science, National Tsing Hua University, Hsinchu 300044, Taiwan
6
Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan
7
Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei 10002, Taiwan
8
Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, Tainan 70101, Taiwan
9
Institute of Biopharmaceutical Sciences, National Yang Ming Chiao Tung University, Taipei 112304, Taiwan
10
Institute of Systems Neuroscience, National Tsing Hua University, Hsinchu 300044, Taiwan
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
‡
These authors contributed equally to this work.
Int. J. Mol. Sci. 2023, 24(1), 612; https://doi.org/10.3390/ijms24010612
Submission received: 31 October 2022 / Revised: 14 December 2022 / Accepted: 27 December 2022 / Published: 29 December 2022
(This article belongs to the Special Issue Molecular and Biological Mechanisms of Longevity)
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Abstract

:
Altered metabolism is a hallmark of aging. The tricarboxylic acid cycle (TCA cycle) is an essential metabolic pathway and plays an important role in lifespan regulation. Supplementation of α-ketoglutarate, a metabolite converted by isocitrate dehydrogenase alpha-1 (idha-1) in the TCA cycle, increases lifespan in C. elegans. However, whether idha-1 can regulate lifespan in C. elegans remains unknown. Here, we reported that the expression of idha-1 modulates lifespan and oxidative stress tolerance in C. elegans. Transgenic overexpression of idha-1 extends lifespan, increases the levels of NADPH/NADP+ ratio, and elevates the tolerance to oxidative stress. Conversely, RNAi knockdown of idha-1 exhibits the opposite effects. In addition, the longevity of eat-2 (ad1116) mutant via dietary restriction (DR) was reduced by idha-1 knockdown, indicating that idha-1 may play a role in DR-mediated longevity. Furthermore, idha-1 mediated lifespan may depend on the target of rapamycin (TOR) signaling. Moreover, the phosphorylation levels of S6 kinase (p-S6K) inversely correlate with idha-1 expression, supporting that the idha-1-mediated lifespan regulation may involve the TOR signaling pathway. Together, our data provide new insights into the understanding of idha-1 new function in lifespan regulation probably via DR and TOR signaling and in oxidative stress tolerance in C. elegans.

1. Introduction

Aging is an irreversible progression accompanied by physiologic function decline progressively and eventually leads to death [1]. Aging and metabolism are highly intertwined [2]. Several mechanisms have been reported to compose a complex regulation of lifespan, and one of the evolutionarily conserved mechanisms is dietary restriction (DR) [3,4]. DR prolongs the lifespan of yeast, C. elegans, Drosophila, and mammals [5]. DR not only affects metabolic rate but also promotes longevity and stress resistance via several signaling pathways [5,6].
Multiple signaling pathways are involved in the mechanism of dietary restriction, such as insulin/IGF-1-like signaling, sirtuin, and target of rapamycin (TOR) signaling [7]. Among those pathways, the TOR pathway is closely linked to DR [8,9]. TOR acts as a nutrient-sensing signaling pathway in which its activity increases when food is abundant and vice versa [7,8,10]. TOR controls protein synthesis by phosphorylating ribosomal protein S6 kinase (S6K) and restrains the activity of transcription factor PHA-4 and FOXO/DAF-16, as well as inhibits autophagy and the expression of stress-related genes [7,9]. Repressing TOR activity by rapamycin is sufficient to extend the lifespan in C. elegans, Drosophila, and mice [11]. Several studies indicated that RNAi silencing TOR or its downstream target S6K can increase lifespan in yeast, Drosophila, and C. elegans [12,13,14].
Metabolic alternation plays an important role during the aging process [15]. The manipulation of genes and metabolites in the tricarboxylic acid (TCA) cycle has been reported to regulate lifespan [16]. Lifespan extension occurs when adding either α-ketoglutarate, succinate, pyruvate, malate, fumarate, or citrate which are all metabolites generated by the TCA cycle, in C. elegans or in Drosophila [17,18,19,20,21,22]. Among these metabolites, α-ketoglutarate supplementation not only prolongs the lifespan in worms and flies but also enhances longevity in mice [23] and may also help in promoting human health [24]. Besides metabolites supplementation, the manipulation of the enzymes in the TCA cycle also can modulate lifespan. The inhibition of gdh-1 and dld-1, which both increase the α-ketoglutarate level, can extend the lifespan in C. elegans [17,25]. Isocitrate dehydrogenase (IDH) is a rate-limiting enzyme which catalyzes isocitrate to become α-ketoglutarate in the TCA cycle [26]. IDH composes three isoenzymes, IDH1, IDH2, and IDH3, which are conserved in eukaryotes. IDH1 and IDH2 are NADP+ dependent, whereas IDH3 is NAD+ dependent. In humans, IDH3 is a heterotetramer containing two alpha subunits (IDH3A), one beat subunit (IDH3B) and one gamma subunit (IDH3G). However, the role of isocitrate dehydrogenase in lifespan regulation remains unknown.
Here, we report that expression of isocitrate dehydrogenase alpha-1 (idha-1), the ortholog of human IHD3A, regulates lifespan and modulates oxidative stress tolerance in C. elegans. Overexpression of idha-1 prolongs lifespan, increases oxidative stress resistance, and elevates the levels of the NADPH/NADP+ ratio. Oppositely, RNAi knockdown of idha-1 displays reduced lifespan, lowered tolerance to oxidative stress, and diminished levels of the NADPH/NADP+ ratio. Mechanistically, the knockdown of idha-1 partially abolishes the longevity in eat-2 (ad1116) dietary restriction (DR) mutant, suggesting idha-1 participates in DR-mediated longevity. Furthermore, the knockdown of idha-1 does not significantly block the enhanced lifespan of the rsks-1 (ok1255) mutant, implying the involvement of TOR signaling. Moreover, the phosphorylation levels of S6 kinase (p-S6K) inversely correlate with the idha-1 expression, supporting that the idha-1-mediated lifespan regulation may involve the TOR signaling pathway. Our study sheds new light on the function of idha-1 in lifespan regulation and in oxidative stress response.

2. Results

2.1. Isocitrate Dehydrogenase Alpha-1 Expression Regulates Lifespan in C. elegans

Supplementation of α-ketoglutarate was reported to extend the lifespan in C. elegans. Hence, we asked whether overexpression of isocitrate dehydrogenase alpha-1 (idha-1), which produces α-ketoglutarate, can enhance lifespan in C. elegans. We generated idha-1 overexpression transgenic worm under its endogenous promoter Pidha-1::idha-1 (also called idha-1 o/e) as well as the control transgenic worm Pidha-1::GFP with GFP under idha-1 endogenous promoter. Our data indicated that Pidha-1::GFP expresses ubiquitously (Figure S1a,b), in the pharynx, body wall muscle, intestine, and nervous system in C. elegans (Figure S1c–e). Overexpression of idha-1 by Pidha-1::idha-1 exhibited an increase in 24.2% in mean lifespan compared with the control Pidha-1::GFP (Figure 1a, Table S1). RT-QPCR analysis also showed a 1.8-fold increase in idha-1 mRNA levels in the idha-1 overexpression worms compared with the control (Figure 1b). Overexpression of idha-1 displayed a 1.75-fold significant increase in IDHA-1 protein levels compared with that in the control (Figure 1c,d). On the other hand, the knockdown of idha-1 by RNAi targeting on either 5’ or 3’ of the idha-1 coding region (named as idha-1 (RNAi) 5’ and idha-1 (RNAi) 3’) in wild type N2 significantly decreased 16.4% and 19.4% in mean lifespan, respectively, compared with the control by empty vector (EV) (Figure 1e, Table S1). RT-QPCR analysis indicated that knockdown of idha-1 by idha-1 (RNAi) 5’ and idha-1 (RNAi) 3’ reduced about 51% and 52% in idha-1 mRNA levels compared with the control (Figure 1f). Knockdown of idha-1 by idha-1 (RNAi) 5’ and idha-1 (RNAi) 3’ both lowered about 68% and 53% in IDHA-1 protein levels compared with the control (Figure 1g,h). Together, the results indicate that expression of idha-1 can modulate lifespan in C. elegans.

2.2. Expression of idha-1 Modulates α-Ketoglutarate Levels

Since idha-1 expression, which produces α-ketoglutarate, can regulate lifespan in C. elegans, we asked whether the expression of idha-1 can modulate α-ketoglutarate levels. Overexpression of idha-1 by Pidha-1::idha-1 significantly increased 1.29-fold in α-ketoglutarate levels compared with the control (Figure 2a). Conversely, the knockdown of idha-1 by idha-1 (RNAi) 5’ and idha-1 (RNAi) 3’ reduced α-ketoglutarate levels to about 0.75- and 0.85-fold, respectively, compared with the control (Figure 2b). The data showed that expression of idha-1, which regulates lifespan, can also modulate α-ketoglutarate levels.

2.3. Expression of idha-1 Manages the Tolerance to Oxidative Stress and Levels of NADPH/NADP+ Ratio

Longevity organism is usually associated with better stress resistance [27,28,29,30]. To examine whether the expression of idha-1 which regulates lifespan is also associated with the tolerance to oxidative stress, we performed an oxidative stress assay with the idha-1 overexpression and knockdown worms and their controls under paraquat-induced oxidative stress. The idha-1 overexpression strain exhibited significantly increased survival under 48 and 72 h of paraquat treatment compared with the control (Figure 3a). On the other hand, the knockdown of idha-1 in N2 significantly reduced the tolerance to oxidative stress compared with the control at 48 and 72 h of the paraquat treatment (Figure 3b). Thus, these data suggest that the expression of idha-1 is associated with the tolerance to oxidative stress by paraquat.
NADPH can reduce glutathione disulfide (GSSG) to glutathione (GSH) to lower free radical levels. The ratio of NADPH/NADP+ level is considered an index for oxidative stress tolerance. The higher the NADPH level, the better the oxidative stress tolerance [28]. Therefore, we measured the NADPH/NADP+ ratio in both idha-1 overexpression and knockdown worms. Overexpression of idha-1 displayed a significant increase in NADPH/NADP+ ratio compared with that in the control (Figure 3c). On the other side, the knockdown of idha-1 showed significantly decreased levels of NADPH/NADP+ ratio compared with the control (Figure 3d). Together, these results reveal that the expression of idha-1, which regulates lifespan, also manages the tolerance to oxidative stress and the levels of NADPH/NADP+ ratio in C. elegans.

2.4. idha-1 Plays A Role in Dietary Restriction Induced Longevity

Reduced fecundity is associated with dietary restriction (DR)-mediated longevity. The long-lived eat-2 (ad1116) mutant, which exhibits reduced pharyngeal pumping rate for lower food intake and is used as a DR model, exhibits a reduction in progeny production. The supplementation of α-ketoglutarate was reported to be unable to further prolong the extended lifespan in the eat-2 (ad1116) mutant, suggesting the longevity by α-ketoglutarate is similar to that by DR. Therefore, we asked whether the long-lived idha-1 overexpression transgenic strain which shows an elevated α-ketoglutarate level displays reduced fecundity. We measured the brood size and found a significant decrease in the number of progenies from day 2 to day 6 in the idha-1 overexpression transgenic line compared with the control (Figure 4a). The average of the total progeny number significantly declines by about 50% in the idha-1 overexpression line compared with the control (Figure 4b). Interestingly, we found the idha-1 mRNA levels are elevated in both the DR-treated worms by fasting and the eat-2 (ad1116) mutant DR model compared with their controls in the GEO data (Figure S2a,b) as well as in the verification by quantitative RT-PCR analysis (Figure S2c). The expression of many other TCA cycle genes is also upregulated in the DR-treated worms by fasting and the eat-2 (ad1116) mutant DR model compared with their controls in the GEO data (Figure S2d,e). The data imply the longevity of idha-1 overexpression may also be involved in DR-induced longevity. Moreover, the knockdown of idha-1 significantly diminished the extended lifespan in eat-2 (ad1116) (Figure 4c, Table S1), which indicates the longevity in eat-2 (ad1116) mutant partially depends on idha-1. Overall, the results support our hypothesis that idha-1 plays a role in dietary restriction-mediated longevity in C. elegans.

2.5. Expression of idha-1 May Regulate Lifespan via Inversely Modulating TOR Signaling

Dietary restriction is known to interact through multiple signaling pathways, such as the insulin/IGF-1 signaling (IIS) pathway, AMP-activated protein kinase (AMPK) pathway and Target of Rapamycin (TOR) pathway [7]. Lifespan extension caused by reducing the IIS pathway and TOR pathway signaling or activating the AMPK pathway is conserved from yeast to mammals [4]. Our finding suggests that idha-1 may be involved in DR-induced longevity. To further study the underlying molecular mechanism of idha-1-mediated lifespan regulation, we examined the lifespan by reducing idha-1 expression in the daf-16 (mu86), aak-2 (gt33), and rsks-1 (ok1255) mutant strains related to IIS, AMPK, and TOR pathways, respectively. Knockdown of idha-1 decreased the lifespan in short-lived daf-16 (mu86) (Figure 5a, Table S1), suggesting that idha-1-mediated lifespan is independent of daf-16. Similarly, the knockdown of idha-1 also reduced the lifespan in the short-lived aak-2 (gt33) mutant (Figure 5b, Table S1), indicating that idha-1-mediated lifespan is independent of aak-2. On the other hand, the knockdown of idha-1 did not significantly shorten the lifespan in long-lived rsks-1 (ok1255) mutant (Figure 5c, Table S1), implying that idha-1-mediated lifespan may depend on reducing TOR signaling.
To strengthen our hypothesis that idha-1-mediated lifespan may depend on lowered TOR signaling, we examined the levels of p-S6K, which is a downstream effector of TOR signaling in both idha-1 overexpression and knockdown strains. The long-lived idha-1 overexpression strain displayed significantly lower p-S6K levels compared with the control (Figure 6a,b), whereas the short-lived idha-1 knockdown lines showed the opposite results with the elevated p-S6K levels compared with the control (Figure 6c,d). Together, the data support the finding that the idha-1-mediated lifespan depends on TOR signaling in C. elegans.

3. Discussion

Despite as an enzyme in the TCA cycle, in this study, we uncover a new function of idha-1 in lifespan regulation and oxidative stress response in C. elegans. Those phenotypes are associated with the levels of NADPH/DADP+ ratio. This is in accordance with the report that Sirt3 can activate mitochondrial isocitrate dehydrogenase 2 to increase NADPH levels in response to caloric restriction in mice [31]. Our previous study also reported that the longevity and oxidative stress resistance by ribose-5-phosphate isomerase knockdown are associated with the levels of NADPH/DADP+ ratio in Drosophila [28]. In addition, we disclose that the mechanism of lifespan regulation by idha-1 could be mediated by dietary restriction and TOR signaling. This is consistent with the previous findings that α-ketoglutarate supplementation prolongs lifespan via inhibiting TOR signaling in C. elegans and Drosophila [17,20]. Moreover, those idha-1-mediated phenotypes not only occur in C. elegans but also are associated with an allele related to isocitrate dehydrogenase in Drosophila by selected breeding [32], suggesting that lifespan regulation and the oxidative stress response by idha-1 may be evolutionary conserved. IDHA-1 is conserved among the Caenorhabditis genus (Figure S3).
The TCA cycle is an energy-producing and metabolism process. Recent studies revealed that the supplementation of TCA cycle metabolites, such as α-ketoglutarate, malate, and fumarate, benefits longevity [17,22]. Not only can the supplementation of certain metabolites in the TCA cycle extend lifespan, but also genetic manipulation of some genes in the TCA cycle can regulate longevity. The knockdown of ogdh-1, sdha-1, and dld-1 can increase α-ketoglutarate levels and also extend the lifespan in C. elegans [17,22,25]. Apart from manipulating the TCA cycle directly, a previous study showed that long-lived rodents tend to upregulate TCA cycle genes or show no decline in TCA cycle function [33,34]. However, among all these studies, no report yet shows single TCA cycle gene overexpression can prolong lifespan. Here, we provide the first new strategy of lifespan extension in nematode by overexpressing the TCA cycle gene idha-1 in C. elegans.
Better oxidative stress tolerance is usually accompanied by a higher antioxidant capacity. Since the elevation of NADPH levels shows better resistance against ROS and free radicals, the NADPH/NADP+ ratio can reflect the tolerance against oxidative stress [28,35]. Here, we found idha-1 overexpression strain exhibits higher NADPH levels, and the knockdown of idha-1 reduced NADPH levels and vice versa. Although the supplementation of α-ketoglutarate has been shown to extend lifespan, it is unable to elevate the oxidative stress tolerance across the species [17,20,23]. The discrepancy could be the additional genetic effect of idha-1 expression, which can modulate NADPH levels. The other possibility is that idha-1 expression may regulate some antioxidant gene expression. Our study provided another new piece of evidence that idha-1 expression modulates oxidative stress tolerance in C. elegans.
Moreover, we discovered that idha-1 overexpression may be linked to the DR-induced longevity phenotype and the reduction in brood size [36]. To investigate the direct relationship between idha-1 and DR, we perform a lifespan assay under DR model conditions and find out whether DR-induced longevity may partially rely on idha-1 expression. DR-induced longevity includes several nutrient-signaling pathways, such as the IIS pathway, AMPK pathway, and TOR pathway [37]. Unraveling the potential mechanism idha-1-mediated lifespan regulation may depend on, we knockdown idha-1 in the mutants of these pathways. The result points out that only the TOR signaling pathway, but not the IIS or AMPK, is responsible for idha-1-mediated lifespan regulation. Together, these results support our hypothesis that idha-1 plays a role in DR-induced longevity through the TOR signaling pathway.
Besides the IIS, AMPK, and TOR pathways, autophagy and sirtuins are also involved in DR-induced longevity [38]. The relationship between the TOR pathway and autophagy among species is well documented. The inhibition of the TOR signaling pathway may cause the activation of autophagy. To further validate the relation between autophagy and idha-1 mediation, we may investigate the molecular changes of some autophagy-related genes, such as bec-1, lgg-1, and sqst-1 [39], or even measure the autophagy flux in the future study.
Aside from autophagy, sirtuins may also involve in idha-1-mediated lifespan regulation. Sirtuins are a family of NAD+-dependent protein deacetylases, which use NAD+ to remove acetyl moieties on histones and proteins [40]. Sirt1 is one of the sirtuins in humans which participates in DR-induced longevity [41]. The activity of Sirt1 accompanies the elevation of NAD+ levels during DR. The orthologue of Sirt1, sir-2.1, can partially regulate DR-induced longevity by activating DAF-16/FOXO [42]. Based on the function of idha-1, which converts NAD+ to NADH, we assumed overexpression of idha-1 may elevate the NADH level. However, we did not observe increased NADH levels when overexpressing idha-1. Surprisingly, we found an elevation of the NAD+ level, which is associated with DR-mediated longevity, upon idha-1 overexpression (Figure S4). A previous study demonstrated that the elevation of NAD+ level accompanies better mitochondrial efficiency and also proposes that the elevation of the NAD+ level is a consequence of DR [43]. As our data show that idha-1 overexpression elevates NAD+ levels and participates in DR-induced longevity, it may imply the involvement of sir-2.1. We may investigate the role of sir-2.1 in idha-1-mediated lifespan regulation in future work.
In summary (Figure 7), we disclose the new role of idha-1 in lifespan regulation and oxidative stress tolerance. Our study demonstrates that idha-1 regulates lifespan by participating in DR-induced longevity inversely through the TOR signaling pathway and modulates oxidative stress tolerance via the levels of NADPH/NADP+ ratio. Increasing amounts of evidence demonstrate that TCA metabolites and enzymes control human physiology and diseases [44,45]. The information from this study will help in the development of α-ketoglutarate as dietary supplementation in promoting human health [24].

4. Materials and Methods

4.1. Generation of RNA Interference Constructs against idha-1

To generate the RNA interference (RNAi) constructs targeting idha-1, we designed two primer sets for two different coding regions of idha-1, one includes exon 2 and exon 3 for 312bp amplified by the primers idha-1 5’ forward: 5’-GCCGCCGATGCC-3’ and idha-1 5’ reverse: 5’-CTCGTGCTCAATTCCAGAG-3’, and the other includes exon 7 and exon 8 for 267bp amplified by the primers idha-1 3’ forward: 5’-GAAGCCTACCTCGACACAG-3’ and idha-1 3’ reverse: 5’-CATGTAACGGAGCATCATG-3’. The amplicons were cloned into the HindIII site in the L4440 vector, and the resultant constructs were named idha-1 (RNAi) 5’ and idha-1 (RNAi) 3’ separately and were transformed into HT115 for the RNAi experiments. The preparation of an RNAi construct containing bacteria for the RNAi experiments was described previously [29,30].

4.2. Generation of idha-1 Overexpression Transgenic Worms

To establish the idha-1 overexpression transgenic construct, we amplified the idha-1 genomic DNA containing 1 kb upstream promoter region and the whole idha-1 genomic DNA to be cloned into L3691 vector by using the primers p1-F idha-1-pstI (5’-TTTCTGCAGTCGAAGTTGTCAAAATCCACGAGA-3’) and p2-R-idha-1-kpnI (5’-TTTGGTACCTCTGAACCCTGGAAACAAAAATATTT-3’). The resultant idha-1 overexpression transgenic construct was named Pidha-1::idha-1. For the control transgenic construct, we cloned the 1 kb idha-1 upstream promoter region into L3691 to express GFP and named the construct Pidha-1::gfp. To generate the idha-1 overexpression transgenic and the control transgenic worms, N2 worms were prepared and synchronized via timed eggs prepared as described previously [30]. The plasmid construct was diluted in ddH2O and injected into young adult worms (4 to 8 eggs) with co-injection vector Psur-5::rfp at a total concentration of 20 ng/µL. The progenies of the injected worms were then screened for RFP expression and with GFP as well for the control and established as the transgenic lines. Worms expressing RFP were picked and transferred onto new plates every 5 days to maintain a stable line and verified by PCR. The control transgenic worm Pidha-1::gfp was used to examine the tissue expression patterns of the GFP driven by the idha-1 promoter.

4.3. Lifespan Analysis and Oxidative Stress Assay

Lifespan assay was performed at 20 °C as described previously [30]. Parent worms were grown to the day-1-adult stage on NGM plates seeded with E. coli strain OP50. Nearly 20 adult worms were transferred to RNAi-containing plates or normal NGM plates for 6 h and picked off. The offspring were synchronized on the plates by timed egg lay assay. After the offspring grew to the L4 stage, 30 worms were picked onto each plate, and at least 120 worms existed in each group per trial. Worms were transferred to new plates and the number of deaths was counted every 2 days until all worms died. For oxidative stress assay, 50 worms were picked onto a new plate containing 10 mM paraquat (1, 1’-Dimethyl-4, 4’-bipyridinium dichloride, Sigma-Aldrich). The number of dead worms was calculated every 24 h. The statistical analyses of lifespan and oxidative stress assay were performed by OASIS 2 and p-values were calculated by log-rank test for lifespan and t-test or one-way ANOVA test for oxidative stress assay.

4.4. Western Blot

Worms with or without specific treatment were collected and lysed by whole-cell extract (WCE) buffer containing 20 mM HEPES (pH 7.5), 75 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, 0.5% Triton X-100, 0.1 mM Na3VO4, 50 mM NaF and protease inhibitor (Roche, Cat#04 693 124 001). The samples with an equal amount of protein were prepared as described previously for the western blot [30]. The primary antibodies used were listed below: IDHA-3A (Genetex, GTX114486), beta-actin (GeneTex, GTX109639), and p-S6K (Cell Signaling, Cat# 9209). The secondary antibody is goat anti-rabbit IgG HRP-conjugate antibodies (JIR, Cat#111-035-003). The resultant NC membrane was then incubated with ECL (Millipore, Cat#WBKLS0500), and detected by ImageQuant (GE Healthcare, LAS 4000 mini). Statistical analysis was performed by ImageJ and p-values were calculated by t-test or one-way ANOVA test.

4.5. RNA Extraction and qRT-PCR

The worms were washed with an M9 buffer three times and collected in a 1.5-mL micro-centrifuge tube. The RNA extraction and RT-QPCR procedures were described previously [30]. The RNA pellet was dissolved by 10 μL DEPC-treated ddH2O and measured the concentration by NanoDrop 2000 (Thermo). The qRT-PCR was performed by using the Step One Plus Real-Time PCR system (ABI). The relative expression levels of genes between different samples were calculated on the basis of ΔΔCt (threshold cycles) values and act-1 was used as the internal control for normalization. Statistical analysis was calculated by t-test or one-way ANOVA test. The primers for idha-1 are the forward primer 5’-GCACGCGAACAAAGTTGGAC-3’ and the reverse primer 5’-CTTCAAGGGAACGGCATGGA-3’. The primers for act-1 are the forward primer 5’-CTCTTGCCCCATCAACCATGA-3’ and the reverse primer 5’-TTGCGGTGAACGATGGATGG-3’.

4.6. Measurement of α-Ketoglutarate Level

The α-ketoglutarate levels were measured by a colorimetric quantification kit (BioVision, Cat# K677-100) for Figure 3c and by a bioluminescent assay-based kit (NADP/NADPH-Glo™ assay; Promega, Cat#G9081) for Figure 3d according to the manufacturer’s protocols. Worms were collected into 1.5-mL centrifuge tubes and homogenized in the extraction buffer provided with the kit. The samples were centrifuged at 13,000 rpm for 5 min and the supernatants were transferred into new labeled tubes. To detect the α-ketoglutarate level, 50 μL samples were transferred into a 96-well ELISA plate. 2 μL of cycling enzyme mix and 48 μL of buffer mix were added in each well and incubated at 37 °C for 30 min. The plate was read under OD 570 nm with an ELISA reader, and the α-ketoglutarate level was calculated according to the standard curve and normalized by protein concentration.

4.7. NADPH/NADP+ and NAD+/NADH Quantification

The NADPH/NADP+ and NAD+/NADH quantifications were measured by using the quantification colorimetric kits (BioVision, Cat#: K337-100 and K347-100). The worms were collected into a 1.5-mL eppendorf tube and homogenized by the extraction buffer. 50 μL of each sample was transferred into a 96-well ELISA plate according to the manufacturer’s protocols. Calculated the ratio according to the standard curve.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms24010612/s1.

Author Contributions

Conceptualization, supervision, wrote the manuscript: H.-D.W.; methodology, Z.-H.L., S.-Y.C., W.-C.S., Y.-H.L., C.-L.S., S.-B.L., Y.-C.L.; resources and intellectual contribution, C.-S.C. and T.-T.C.; writing and editing, Z.-H.L., S.-Y.C., W.-C.S. All authors have read and agreed to the published version of the manuscript.

Funding

The research was funded by National Science and Technology Council grant number 106-2311-B-007-006-MY2, 108-2311-B-007-007-, 109-2311-B-007-002-, 110-2320-B-007-003-, 111-2320-B-007-006-MY3 and by National Tsing Hua University grant number 111FMDSE1.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We would like to thank Ao-Lin Hsu, Yi-Chun Wu, Chiou-Hwa Yuh, Chun-Liang Pan, Bi-Tzen Juang, and the Taiwan C. elegans core facility funded by National Science and Technology Council (NSTC) Taiwan for the assistance.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Expression of idha-1 regulates lifespan in C. elegans. (a) idha-1 overexpression (red line, Pidha-1::idha-1, mean lifespan: 15.9 days, n = 112) exhibits a 24.2% increase in mean lifespan extension (p < 0.0001, log-rank test) compared with the control (black line, Pidha-1::gfp, mean lifespan: 12.8 days, n = 109). N2 (blue dotted line, mean lifespan: 16.4 days, n = 115). (b) The idha-1 overexpression strain (red bar) showed a nearly 1.8-fold increase in idha-1 mRNA levels compared with the control (grey bar) normalized with act-1. (** p < 0.01, t-test). (c,d) The elevated IDHA-1 protein levels were detected in idha-1 overexpression worms (idha-1 o/e). (* p < 0.05, t-test). (e) Knockdown of idha-1 (blue line, N2 idha-1 (RNAi) 5’, mean lifespan: 16.8 days, n = 173; and yellow line, N2 idha-1 (RNAi) 3’, mean lifespan: 16.2 days, n = 167) reduces mean lifespan (−16.4% and −19.4% respectively, both p < 0.0001, log-rank test) compared with the control (black line, N2 EV, mean lifespan: 20.1 days, n = 169). (f) The idha-1 knockdown strains (blue bar, N2 idha-1 (RNAi) 5’ and yellow bar, N2 idha-1 (RNAi) 3’) both decreased 0.5-fold in idha-1 mRNA levels compared with the control (black bar, N2 EV) normalized with act-1. (* p < 0.05, one-way ANOVA test). (g,h) The decreased IDHA-1 protein levels were detected by the knockdown of idha-1 (*** p < 0.001 and * p < 0.05 respectively, one-way ANOVA test). Each bar represents mean ± SD.
Figure 1. Expression of idha-1 regulates lifespan in C. elegans. (a) idha-1 overexpression (red line, Pidha-1::idha-1, mean lifespan: 15.9 days, n = 112) exhibits a 24.2% increase in mean lifespan extension (p < 0.0001, log-rank test) compared with the control (black line, Pidha-1::gfp, mean lifespan: 12.8 days, n = 109). N2 (blue dotted line, mean lifespan: 16.4 days, n = 115). (b) The idha-1 overexpression strain (red bar) showed a nearly 1.8-fold increase in idha-1 mRNA levels compared with the control (grey bar) normalized with act-1. (** p < 0.01, t-test). (c,d) The elevated IDHA-1 protein levels were detected in idha-1 overexpression worms (idha-1 o/e). (* p < 0.05, t-test). (e) Knockdown of idha-1 (blue line, N2 idha-1 (RNAi) 5’, mean lifespan: 16.8 days, n = 173; and yellow line, N2 idha-1 (RNAi) 3’, mean lifespan: 16.2 days, n = 167) reduces mean lifespan (−16.4% and −19.4% respectively, both p < 0.0001, log-rank test) compared with the control (black line, N2 EV, mean lifespan: 20.1 days, n = 169). (f) The idha-1 knockdown strains (blue bar, N2 idha-1 (RNAi) 5’ and yellow bar, N2 idha-1 (RNAi) 3’) both decreased 0.5-fold in idha-1 mRNA levels compared with the control (black bar, N2 EV) normalized with act-1. (* p < 0.05, one-way ANOVA test). (g,h) The decreased IDHA-1 protein levels were detected by the knockdown of idha-1 (*** p < 0.001 and * p < 0.05 respectively, one-way ANOVA test). Each bar represents mean ± SD.
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Figure 2. Expression of idha-1 mediates α-ketoglutarate levels. (a) Overexpression of idha-1 (red bar) significantly increases α-ketoglutarate levels compared with the control (grey bar). (** p < 0.01, t-test). (b) Knockdown of idha-1 (blue bar, N2 idha-1 (RNAi) 5’ and yellow bar, N2 idha-1 (RNAi) 3’) shows lowered α-ketoglutarate levels compared with the control (black bar, N2 EV). (* p < 0.05, one-way ANOVA test). Each bar represents mean ± SD, n = 3.
Figure 2. Expression of idha-1 mediates α-ketoglutarate levels. (a) Overexpression of idha-1 (red bar) significantly increases α-ketoglutarate levels compared with the control (grey bar). (** p < 0.01, t-test). (b) Knockdown of idha-1 (blue bar, N2 idha-1 (RNAi) 5’ and yellow bar, N2 idha-1 (RNAi) 3’) shows lowered α-ketoglutarate levels compared with the control (black bar, N2 EV). (* p < 0.05, one-way ANOVA test). Each bar represents mean ± SD, n = 3.
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Figure 3. Expression of idha-1 modulates the tolerance to oxidative stress and NADPH/NADP+ levels. (a) Overexpression of idha-1 (red dot, n = 263) significantly increases oxidative stress tolerance after 48 and 72 h of paraquat treatment (** p< 0.01, t-test) compared with the control (black dot, n = 252). (b) Knockdown of idha-1 (blue dot, N2 idha-1 (RNAi) 5’, n = 164; and yellow dot, N2 idha-1 (RNAi) 3’, n = 158) significantly reduces the tolerance to oxidative stress compared with the control (black dot, N2 EV, n = 157) after 24-, 48- and 72-h treatment (** p < 0.01, **** p < 0.0001, one-way ANOVA test). Each dot represents mean ± SD, n = 3. (c) Overexpression of idha-1 (red bar) significantly increases the NADPH/NADP+ ratio levels compared with the control (grey bar). (** p < 0.01, t-test). (d) Knockdown of idha-1 (blue bar, N2 idha-1 (RNAi) 5’ and yellow bar, N2 idha-1 (RNAi) 3’) significantly decreases the NADPH/NADP+ ratio levels compared with the control (black bar, N2 EV). (*** p < 0.001 and * p < 0.05, one-way ANOVA test). Each bar represents mean ± SD, n = 3.
Figure 3. Expression of idha-1 modulates the tolerance to oxidative stress and NADPH/NADP+ levels. (a) Overexpression of idha-1 (red dot, n = 263) significantly increases oxidative stress tolerance after 48 and 72 h of paraquat treatment (** p< 0.01, t-test) compared with the control (black dot, n = 252). (b) Knockdown of idha-1 (blue dot, N2 idha-1 (RNAi) 5’, n = 164; and yellow dot, N2 idha-1 (RNAi) 3’, n = 158) significantly reduces the tolerance to oxidative stress compared with the control (black dot, N2 EV, n = 157) after 24-, 48- and 72-h treatment (** p < 0.01, **** p < 0.0001, one-way ANOVA test). Each dot represents mean ± SD, n = 3. (c) Overexpression of idha-1 (red bar) significantly increases the NADPH/NADP+ ratio levels compared with the control (grey bar). (** p < 0.01, t-test). (d) Knockdown of idha-1 (blue bar, N2 idha-1 (RNAi) 5’ and yellow bar, N2 idha-1 (RNAi) 3’) significantly decreases the NADPH/NADP+ ratio levels compared with the control (black bar, N2 EV). (*** p < 0.001 and * p < 0.05, one-way ANOVA test). Each bar represents mean ± SD, n = 3.
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Figure 4. idha-1 plays a role in dietary restriction-induced longevity. (a) Overexpression of idha-1 (red dot) significantly decreases progeny number between day 2 to day 6 compared with the control (black dot). Each dot plot represents mean ± SD, n = 3 (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, t-test) (b) Total brood size is significantly diminished in idha-1 overexpression strain (red bar) compared with the control worm (grey bar). Each dot plot represents mean ± SD, n = 5 (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, t-test). (c) Knockdown of idha-1 in eat-2 (ad1116) longevity mutant (blue dotted line, mean lifespan: 20.8 days, n = 120) partially abolishes the extended lifespan in eat-2 (ad1116) longevity mutant (black dotted line, mean lifespan: 25.7 days, n = 113) compared with the control (black solid line, mean lifespan: 20.1 days, n = 169). Knockdown of idha-1 in N2 (blue solid line, mean lifespan: 16.8 days, n = 193) displays a reduced lifespan compared with the control (black solid line). (p < 0.0001, log-rank test).
Figure 4. idha-1 plays a role in dietary restriction-induced longevity. (a) Overexpression of idha-1 (red dot) significantly decreases progeny number between day 2 to day 6 compared with the control (black dot). Each dot plot represents mean ± SD, n = 3 (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, t-test) (b) Total brood size is significantly diminished in idha-1 overexpression strain (red bar) compared with the control worm (grey bar). Each dot plot represents mean ± SD, n = 5 (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, t-test). (c) Knockdown of idha-1 in eat-2 (ad1116) longevity mutant (blue dotted line, mean lifespan: 20.8 days, n = 120) partially abolishes the extended lifespan in eat-2 (ad1116) longevity mutant (black dotted line, mean lifespan: 25.7 days, n = 113) compared with the control (black solid line, mean lifespan: 20.1 days, n = 169). Knockdown of idha-1 in N2 (blue solid line, mean lifespan: 16.8 days, n = 193) displays a reduced lifespan compared with the control (black solid line). (p < 0.0001, log-rank test).
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Figure 5. The idha-1-mediated lifespan may depend on TOR signaling but not on insulin or AMPK signaling pathway. (a) Knockdown of idha-1 in N2 (blue solid line, mean lifespan: 11.3 days, n = 141) (p < 0.0001, log-rank test) shortened lifespan compared with the control (black solid line, mean lifespan: 14.7 days, n = 139). The shortened lifespan caused by daf-16 mutation (mu86) (black dotted line, mean lifespan: 10.9 days, n = 121) (p < 0.0001, log-rank test) can be further reduced by idha-1 knockdown (blue dotted line, mean lifespan: 9.0 days, n = 120) (p < 0.0001, log-rank test). (b) The shortened lifespan caused by aak-2 mutation (gt33) (black dotted line, mean lifespan: 12.2 days, n = 141) (p < 0.0001, log-rank test) can be further decreased by idha-1 knockdown (blue dotted line, mean lifespan: 10.8 days, n = 117) (p < 0.001, log-rank test). Knockdown of idha-1 in N2 (blue solid line, mean lifespan: 12.2 days, n = 141) (p < 0.0001, log-rank test) shortened lifespan compared with the control (black solid line, mean lifespan: 17.2 days, n = 139). (c) Knockdown of idha-1 in long-lived rsks-1 mutant strain (ok1255) (blue dotted line, mean lifespan: 16.3 days, n = 122) (p = 0.052, log-rank test) cannot significantly abolish the extended lifespan caused by rsks-1 mutation (black dotted line, mean lifespan: 18.1 days, n = 122) (p < 0.05, log-rank test). Knockdown of idha-1 in N2 (blue solid line, mean lifespan: 12.1 days, n = 119) (p < 0.0001, log-rank test) significantly shortened lifespan compared with the control (black solid line, mean lifespan: 16.4 days, n = 118).
Figure 5. The idha-1-mediated lifespan may depend on TOR signaling but not on insulin or AMPK signaling pathway. (a) Knockdown of idha-1 in N2 (blue solid line, mean lifespan: 11.3 days, n = 141) (p < 0.0001, log-rank test) shortened lifespan compared with the control (black solid line, mean lifespan: 14.7 days, n = 139). The shortened lifespan caused by daf-16 mutation (mu86) (black dotted line, mean lifespan: 10.9 days, n = 121) (p < 0.0001, log-rank test) can be further reduced by idha-1 knockdown (blue dotted line, mean lifespan: 9.0 days, n = 120) (p < 0.0001, log-rank test). (b) The shortened lifespan caused by aak-2 mutation (gt33) (black dotted line, mean lifespan: 12.2 days, n = 141) (p < 0.0001, log-rank test) can be further decreased by idha-1 knockdown (blue dotted line, mean lifespan: 10.8 days, n = 117) (p < 0.001, log-rank test). Knockdown of idha-1 in N2 (blue solid line, mean lifespan: 12.2 days, n = 141) (p < 0.0001, log-rank test) shortened lifespan compared with the control (black solid line, mean lifespan: 17.2 days, n = 139). (c) Knockdown of idha-1 in long-lived rsks-1 mutant strain (ok1255) (blue dotted line, mean lifespan: 16.3 days, n = 122) (p = 0.052, log-rank test) cannot significantly abolish the extended lifespan caused by rsks-1 mutation (black dotted line, mean lifespan: 18.1 days, n = 122) (p < 0.05, log-rank test). Knockdown of idha-1 in N2 (blue solid line, mean lifespan: 12.1 days, n = 119) (p < 0.0001, log-rank test) significantly shortened lifespan compared with the control (black solid line, mean lifespan: 16.4 days, n = 118).
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Figure 6. Expression of idha-1 negatively modulates the phosphorylated S6K levels. The level of phosphorylated S6K (p-S6K), a TOR activity indicator, in each strain was determined and normalized with β-actin level. (a,b) Long-lived idha-1 overexpression strain (red bar, idha-1 o/e) displays significantly reduced p-S6K levels compared with the control (grey bar) (** p < 0.01, t test). Each bar represents mean ± SD, n = 4. (c,d) Short-lived idha-1 knockdown strains (blue bar, N2 idha-1 (RNAi) 5’; and yellow bar, N2 idha-1 (RNAi) 3’) exhibit significantly increased p-S6K levels compared with the control (black bar, N2 EV) (* p < 0.05, t test). Each bar represents mean ± SD, n = 3.
Figure 6. Expression of idha-1 negatively modulates the phosphorylated S6K levels. The level of phosphorylated S6K (p-S6K), a TOR activity indicator, in each strain was determined and normalized with β-actin level. (a,b) Long-lived idha-1 overexpression strain (red bar, idha-1 o/e) displays significantly reduced p-S6K levels compared with the control (grey bar) (** p < 0.01, t test). Each bar represents mean ± SD, n = 4. (c,d) Short-lived idha-1 knockdown strains (blue bar, N2 idha-1 (RNAi) 5’; and yellow bar, N2 idha-1 (RNAi) 3’) exhibit significantly increased p-S6K levels compared with the control (black bar, N2 EV) (* p < 0.05, t test). Each bar represents mean ± SD, n = 3.
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Figure 7. The mechanism of idha-1-mediated lifespan regulation and oxidative stress response in C. elegans. DR may induce idha-1 upregulation. Overexpression of idha-1 results in increased levels of α-ketoglutarate and NAD+. In addition, idha-1 overexpression also leads to elevated levels of NADPH/NADH+ ratio which increases oxidative stress tolerance. Moreover, idha-1 overexpression may negatively regulate TOR signaling to extend lifespan in C. elegans.
Figure 7. The mechanism of idha-1-mediated lifespan regulation and oxidative stress response in C. elegans. DR may induce idha-1 upregulation. Overexpression of idha-1 results in increased levels of α-ketoglutarate and NAD+. In addition, idha-1 overexpression also leads to elevated levels of NADPH/NADH+ ratio which increases oxidative stress tolerance. Moreover, idha-1 overexpression may negatively regulate TOR signaling to extend lifespan in C. elegans.
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Lin, Z.-H.; Chang, S.-Y.; Shen, W.-C.; Lin, Y.-H.; Shen, C.-L.; Liao, S.-B.; Liu, Y.-C.; Chen, C.-S.; Ching, T.-T.; Wang, H.-D. Isocitrate Dehydrogenase Alpha-1 Modulates Lifespan and Oxidative Stress Tolerance in Caenorhabditis elegans. Int. J. Mol. Sci. 2023, 24, 612. https://doi.org/10.3390/ijms24010612

AMA Style

Lin Z-H, Chang S-Y, Shen W-C, Lin Y-H, Shen C-L, Liao S-B, Liu Y-C, Chen C-S, Ching T-T, Wang H-D. Isocitrate Dehydrogenase Alpha-1 Modulates Lifespan and Oxidative Stress Tolerance in Caenorhabditis elegans. International Journal of Molecular Sciences. 2023; 24(1):612. https://doi.org/10.3390/ijms24010612

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Lin, Zhi-Han, Shun-Ya Chang, Wen-Chi Shen, Yen-Hung Lin, Chiu-Lun Shen, Sin-Bo Liao, Yu-Chun Liu, Chang-Shi Chen, Tsui-Ting Ching, and Horng-Dar Wang. 2023. "Isocitrate Dehydrogenase Alpha-1 Modulates Lifespan and Oxidative Stress Tolerance in Caenorhabditis elegans" International Journal of Molecular Sciences 24, no. 1: 612. https://doi.org/10.3390/ijms24010612

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