Mitochondrial Aconitase 2 as a Candidate Node Linking Redox Stress, Citrate Partitioning, and Insulin Resistance in Type 2 Diabetes

We propose a testable hypothesis: nutrient excess and mitochondrial redox disturbances such as elevating mitochondrial NADH supply and membrane potential, potentially increasing electron leakage, secondary ROS/RNS production may then impair ACO2 activity. reducing citrate clearance and potentially increasing the availability of citrate for export through the mitochondrial citrate carrier SLC25A1. In the cytosol, citrate can be converted by ATP-citrate lyase (ACLY) to acetyl-CoA, which may supports lipid synthesis and protein acetylation. In some contexts, these processes could contribute to lipid-induced insulin resistance and inflammatory gene expression.

1. Introduction

Type 2 diabetes (T2D) is characterized by hyperglycemia, insulin resistance, and chronic low-grade inflammation. Mitochondria play a central role in the regulation of glucose, fatty-acid, and amino-acid metabolism, as well as cellular redox balance and energy homeostasis. Although mitochondrial dysfunction in metabolic disease is commonly discussed in terms of impaired ATP production, alterations in metabolic flux and carbon partitioning may be equally important.

One potential regulator of these processes is mitochondrial aconitase 2 (ACO2), which catalyzes the reversible isomerization of citrate to isocitrate via cis-aconitate in the tricarboxylic acid (TCA) cycle. ACO2 contains a redox-sensitive iron–sulfur cluster and is susceptible to oxidative inactivation. Its impairment may therefore alter citrate metabolism under conditions of persistent metabolic stress.

This review evaluates the evidence for each component of the proposed mechanism, distinguishes established findings from unresolved causal links, and identifies experiments needed to determine whether ACO2-dependent citrate handling contributes meaningfully to the development of metabolic disease.

We propose a testable hypothesis: nutrient excess and mitochondrial redox disturbances such as elevating mitochondrial NADH supply and membrane potential, potentially increasing electron leakage, secondary ROS/RNS production may then impair ACO2 activity. reducing citrate clearance and potentially increasing the availability of citrate for export through the mitochondrial citrate carrier SLC25A1. In the cytosol, citrate can be converted by ATP-citrate lyase (ACLY) to acetyl-CoA, which may supports lipid synthesis and protein acetylation. In some contexts, these processes could contribute to lipid-induced insulin resistance and inflammatory gene expression.

2. Redox state, reductive stress, and Aconitase lesion

Mitochondria generate superoxide and H₂O₂ at several sites, and the amount produced depends on the reduction state of electron carriers and on membrane potential. When nutrient supply outpaces ATP demand, NADH accumulates and electron flux through complex I and the CoQ pool becomes "congested," a state described as reductive stress. Yan (2014) argued that chronic hyperglycaemia produces NADH overload that increases electron leakage and ROS, so that reductive stress precedes oxidative stress [1]. Aconitase (ACO2) catalyses the isomerisation of citrate to isocitrate via cis-aconitate in mitochondria in TCA cycling, using a [4Fe–4S] cluster in which one iron is not coordinated by a cysteine and binds substrate. Because ACO2 is not an NADH-dependent enzyme, NADH accumulation itself does not establish ACO2 inhibition; the proposed link is indirect and depends on ROS/RNS-mediated cluster injury.

The exposed iron makes the enzyme vulnerable to superoxide, peroxynitrite, and nitric oxide, converting the cluster to an inactive [3Fe–4S] form in some contexts with iron release which is reductive stress–associated, ROS-mediated ACO2 inhibition [2]. Gardner and Fridovich established aconitase as a sensitive biochemical indicator of superoxide [2,3], and its loss has been used as a marker of mitochondrial oxidative damage; for example, Yan et al. found aconitase to be a prominent oxidation target in ageing flies [4].

The consequences of inactivation are more complicated than often assumed. Scandroglio et al. studied isolated rat tissue mitochondria and found that oxygen consumption fell only after aconitase inhibition exceeded a threshold ranging from 63 to 98% across tissues. The inhibition can subsequently be reversed under appropriate conditions, indicating that aconitase can function as a dynamic redox-sensitive metabolic sensor rather than merely being irreversibly destroyed. Heart and brain showed the highest flux control, and inhibition lowered membrane potential and H₂O₂ production in those tissues. The authors interpret inactivation as a possible way to limit superoxide and H₂O₂ formation, and note the enzyme can be reactivated by reductants and iron [5]. Inactivation is therefore better viewed as a regulated, potentially reversible state than as irreversible damage. Therefore, the functional assay of ACO2  could be considered in  three states, revisible, irrevisible and low abundance. Human genetics adds some perspective: biallelic ACO2 mutations cause an infantile cerebellar–retinal degeneration [6], showing that the enzyme is essential in high-energy neural tissue.

3. Citrate export, ACLY, and acetyl-CoA signaling

Citrate leaves mitochondria in exchange for malate or other dicarboxylates through SLC25A1, the citrate carrier (CIC). The carrier is regulated by inflammatory stimuli. Infantino and colleagues reported induction of SLC25A1 expression and activity in LPS-stimulated macrophages, which required for part of the inflammatory response [7,8]. Williams and O'Neill review citrate as a node in innate-immune metabolic reprogramming. In humans, SLC25A1 deficiency causes combined D-2- and L-2-hydroxyglutaric aciduria with severe neurological disease [9,10], again showing physiological importance without addressing partial modulation.

Two questions follow: first, does ACO2 inhibition increase citrate accumulation under nutrient-replete conditions; second, does it increase SLC25A1-mediated citrate export independently of, or in addition to, increased substrate availability? A 2026 Cell study demonstrated that citrate clearance is a major function of ACO2 and that disrupting citrate catabolism causes intracellular citrate accumulation and cellular stress. In vivo ACO2 deficiency produced substantial citrate accumulation and tissue pathology [11]. The second question remains particularly interesting because the transportation is not only dependent on the concentration of substrate, but also the state of the carrier, as inflammatory signaling, metabolic state, and possibly post-translational regulation could alter carrier activity independently of substrate supply. Donnelly et al. showed that mitochondrial superoxide-induced aconitase inhibition promoted citrate export and cytosolic acetyl-CoA-dependent histone acetylation in a mitohormesis model; this study did not establish a lipogenic or insulin-resistance endpoint (12).

In the cytosol, ATP-citrate lyase (ACLY) cleaves exported citrate to acetyl-CoA and oxaloacetate. Acetyl-CoA is a substrate for fatty-acid and cholesterol synthesis and the sole acetyl donor for protein acetylation, so its compartmental availability couples metabolism to chromatin. ACLY-derived acetyl-CoA supports histone acetylation in a nutrient-dependent manner [13].

Pharmacological inhibition of the pathway has shown promise in metabolic disease. The SLC25A1 inhibitor CTPI-2 reversed steatosis, reduced inflammatory macrophage infiltration in liver and adipose tissue, curbed diet-induced obesity, and normalised glucose intolerance in preclinical non-alcoholic fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NASH) models [14]. Inhibiting ACLY improved NASH, fibrosis, and dyslipidaemia in mouse models [15], and the ACLY inhibitor bempedoic acid reduced cardiovascular events in statin-intolerant patients in clear outcomes [16].

4. The citrate–acetyl-CoA pathway and lipid-induced insulin resistance and inflammation

Cytosol citrate facilitates acetyl-CoA production and consquencially, malonyl-CoA and fatty acyl-CoA synthesis. While glucose simultaneously overloading will promotes glycolysis and glycerol-3-phosphate production, the two streams converge to synthesize diacylglycerol (DAG). The leading mechanistic account of lipid-induced insulin resistance holds that ectopic lipid accumulation raises DAG, activating novel PKC isoforms that inhibit insulin signalling. In skeletal muscle, DAG–PKCθ phosphorylates IRS-1 on serine residues, impairing PI3K signalling and GLUT4 translocation; PKCθ knockout mice are protected from fat-induced resistance [17]. Human studies using lipid infusion and lipidomics support a role for DAG–PKCθ in muscle [18]. In the liver, DAG activates PKCε, which phosphorylates the insulin receptor at Thr1160 and blocks signalling [19]. Ceramides provide a parallel route, and the athlete's paradox, where trained muscle holds high lipid yet remains insulin sensitive, shows that content alone is not sufficient to predict resistance [20].

Human and rodent evidence supports a link between lipid oversupply, mitochondrial H₂O₂ emission, and insulin resistance. Anderson et al. found that high-fat feeding raised mitochondrial H₂O₂ emission and shifted redox state, and that mitochondrial-targeted antioxidants prevented insulin resistance in rodents [21]. Against a simple "ROS cause resistance" reading, Hoehn et al. showed that insulin resistance can act as a protective response to excess mitochondrial ROS [22].

Senescent cells secrete cytokines, chemokines, and proteases collectively termed the SASP [23]. Mitochondrial dysfunction induces a distinct senescence-associated secretory profile, and mitochondrial DNA released after apoptotic stress activates innate signalling to drive the SASP [24]. Senescent-cell clearance improves metabolic function in obese mice, which links senescence to metabolic disease.

Martini and colleagues, in Nature (2026), added a metabolic layer: in senescent cells the mitochondrial pyruvate–citrate–acetyl-CoA axis is upregulated, increasing acetyl-CoA for histone acetylation at SASP genes, and SLC25A1 inhibition reduces that acetylation [25]. Inhibiting SLC25A1 or ACLY suppressed SASP expression without affecting cell-cycle arrest, and pharmacological SLC25A1 inhibition reduced systemic inflammation and extended healthspan in aged mice [25].

5. Knowledge gaps and conclusion

We propose that persistent nutrient excess may promote mitochondrial NADH accumulation and redox imbalance. Secondary ROS and RNS production may then impair the ACO2 [4Fe–4S] cluster, reducing citrate-to-isocitrate conversion. If mitochondrial citrate synthesis and SLC25A1-mediated export remain sufficient, ACO2 impairment could increase cytosolic citrate availability. ACLY-dependent conversion of exported citrate to acetyl-CoA may then support de novo lipogenesis and acetyl-CoA-dependent protein acetylation. In hepatocytes, lipogenesis could contribute to signaling-active DAG formation, PKCε activation, insulin-receptor inhibition, and impaired hepatic insulin action. In other cellular contexts, including senescent cells, the same citrate–acetyl-CoA axis may instead support adaptive or inflammatory chromatin remodeling. Thus, ACO2-dependent citrate handling may represent a context-dependent node linking mitochondrial redox state, carbon partitioning, lipid metabolism, and gene regulation.

Nevertheless, several critical questions remain unresolved. It has not been established that ACO2 impairment precedes increased citrate export in nutrient-excess models, that this pathway makes a substantial contribution to DAG accumulation in relevant tissues, or that it is a major causal driver of human T2D. Moreover, partial ACO2 inhibition may reduce mitochondrial oxidant production under some conditions, suggesting that its effects may be adaptive in the short term but maladaptive when persistent.

The next step is to test the proposed sequence directly. Time-resolved experiments should measure ACO2 activity, mitochondrial and cytosolic citrate, SLC25A1-dependent transport, isotope-traced carbon flux into lipids, DAG molecular species, PKC activation, and insulin signaling. These studies should be conducted in relevant tissues and disease models, followed by human investigations to assess the pathway's clinical significance.

The available evidence supports investigating mitochondrial citrate handling as a potential metabolic and epigenetic regulatory node. Whether ACO2 dysfunction is an initiating cause, a contributing factor, or a compensatory response in T2D remains an open scientific question.

Statement

HN.J. and B.X. contributed equally to this work. The authors declare no competing interests.

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References

1.Yan LJ. Pathogenesis of chronic hyperglycemia: from reductive stress to oxidative stress. J Diabetes Res.2014;2014:137919.

2.Gardner PR, Fridovich I. Superoxide sensitivity of the Escherichia coli aconitase. J Biol Chem. 1991;266:19328–19333.

3.Hausladen A, Fridovich I. Superoxide and peroxynitrite inactivate aconitases, but nitric oxide does not. J Biol Chem. 1994;269:29405–29408.

4.Yan LJ, Levine RL, Sohal RS. Oxidative damage during aging targets mitochondrial aconitase. Proc Natl Acad Sci USA. 1997;94:11168–11172.

5.Scandroglio F, Tórtora V, Radi R, Castro L. Metabolic control analysis of mitochondrial aconitase: influence over respiration and mitochondrial superoxide and hydrogen peroxide production. Free Radic Res. 2014;48:684–693.

6.Spiegel R, et al. Infantile cerebellar-retinal degeneration associated with a mutation in mitochondrial aconitase, ACO2. Am J Hum Genet. 2012;90:518–523.

7.Palmieri EM, et al. Nitric oxide orchestrates metabolic rewiring in M1 macrophages by targeting aconitase 2 and pyruvate dehydrogenase. Nat Commun. 2020;11:698.

8.Infantino V, et al. The mitochondrial citrate carrier: a new player in inflammation. Biochem J. 2011;438:433–436.

9.Williams NC, O'Neill LAJ. A role for the Krebs cycle intermediate citrate in metabolic reprogramming in innate immunity and inflammation. Front Immunol. 2018;9:141.

10. Nota B, et al. Deficiency in SLC25A1, encoding the mitochondrial citrate carrier, causes combined D-2- and L-2-hydroxyglutaric aciduria. Am J Hum Genet. 2013;92:627–631.

11. Xie A et al. Citrate clearance is a major function of aconitase 2 in the canonical TCA cycle. Cell. 2026 April 30; 189(9): 2684–2699.

12. Donnelly MP et al.  Mitochondrial superoxide-induced mitohormesis is mediated by citrate and cardioprotective. Sci Adv. 2026 Sep 4;12(36):eaef8132.

13.Wellen KE, et al. ATP-citrate lyase links cellular metabolism to histone acetylation. Science. 2009;324:1076–1080. 

14. Tan M, et al. Inhibition of the mitochondrial citrate carrier, Slc25a1, reverts steatosis, glucose intolerance, and inflammation in preclinical models of NAFLD/NASH. Cell Death Differ. 2020;27:2143–2157.

15. Morrow MR, et al. Inhibition of ATP-citrate lyase improves NASH, liver fibrosis, and dyslipidemia. Cell Metab.2022;34:919–936.

16. Ray KK, et al. Safety and efficacy of bempedoic acid to reduce cardiovascular events and diabetes: in the clear outcomes randomised trial. Lancet Diabetes Endocrinol. 2024 Jan;12(1):19-28

17. Kim JK, et al. PKC-θ knockout mice are protected from fat-induced insulin resistance. J Clin Invest.2004;114:823–827.

18. Szendroedi J, et al. Role of diacylglycerol activation of PKCθ in lipid-induced muscle insulin resistance in humans. Proc Natl Acad Sci USA. 2014;111:9597–9602.

19. Petersen MC, et al. Insulin receptor Thr1160 phosphorylation mediates lipid-induced hepatic insulin resistance. J Clin Invest. 2016;126:4361–4371.

20. Amati F, et al. Skeletal muscle triglycerides, diacylglycerols, and ceramides in insulin resistance: another paradox in endurance-trained athletes? Diabetes. 2011;60:2588–2597.

21. Anderson EJ, et al. Mitochondrial H₂O₂ emission and cellular redox state link excess fat intake to insulin resistance in both rodents and humans. J Clin Invest. 2009;119:573–581.

22. Hoehn KL, et al. Insulin resistance is a cellular antioxidant defense mechanism. Proc Natl Acad Sci USA.2009;106:17787–17792.

23. Wiley CD, et al. Mitochondrial dysfunction induces senescence with a distinct secretory phenotype. Cell Metab.2016;23:303–314.

24. Victorelli S, et al. Apoptotic stress causes mtDNA release during senescence and drives the SASP. Nature.2023;622:627–636.

25. Martini H, Birch J, Marques FDM, et al. Mitochondrial metabolism and epigenetic crosstalk drive SASP. Nature.2026;656(8129):980–992.

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