The Master Switch of Metabolic Disease and Inflammaging: Excess ROS Inhibits Aconitase, Leading to Acetyl-CoA Overproduction, Which in Turn Drives Both Insulin Resistance and Inflammaging — A Unified Mechanistic Hypothesis Linking High-Calorie, High-Carbohydrate, High-Fat Diet, Physical Inactivity, and Micronutrient Deficiency to Metabolic Disease and Pathological Aging

An Exploratory Framework Based on Integrated Intervention with Calorie control，MRD, MLCT, and Micronutrients

Abstract In recent years, the treatment of metabolic diseases (type 2 diabetes, fatty liver disease) and aging-related diseases (arteriosclerosis, neurodegeneration) has gradually fallen into a dilemma of "fighting separate battles." A large body of clinical and basic research suggests that these conditions may share the same upstream origin at the molecular level. A 2026 study published in Nature revealed an SLC25A1-mediated mitochondrial citrate efflux and its driven SASP inflammatory pathway in senescent cells; while insulin resistance research has accumulated substantial evidence that cytosolic acetyl-CoA blocks insulin signaling through the DAG-PKCθ pathway. We attempt to build a bridge between these two independent findings: a high-calorie, high-carbohydrate, high-fat diet leads to an elevated NADH/NAD⁺ ratio, generating "reductive stress." Excess ROS first attacks aconitase, causing mitochondrial citrate accumulation and efflux via SLC25A1, which then diverges into two pathological branches: insulin resistance and inflammaging. Based on this hypothesis, this article proposes an integrated dietary intervention strategy centered on protecting aconitase activity (caloric control, MRD, MLCT, resistant starch, and micronutrient supplementation), and explores its possible path from mechanistic validation to industrial translation.

I. Clinical Dilemma: Why Do Glucose Lowering and Anti-Inflammation Often "Fight Separate Battles"?

In the current diagnostic and therapeutic system, type 2 diabetes belongs to endocrinology, arteriosclerosis to cardiology, and Alzheimer's disease to neurology. They are handled separately in clinical practice, but the frequency with which they co-occur in the same individual is too high to be explained by "coincidence."

Behind this "same disease, different departments" pattern is our inertial dependence on disease classification, rather than a common directional understanding of the etiology. In recent years, increasing evidence suggests:

• Insulin      resistance is not only the core pathology of diabetes but also an      important driver of NAFLD, PCOS, and arteriosclerosis;

• Chronic      low-grade inflammation (inflammaging) is not only the background of      multimorbidity in the elderly but also an independent risk factor for the      development and progression of type 2 diabetes;

• The      simultaneous appearance of these two pathological pathways in the same      metabolic stress environment suggests that they may share a common      upstream origin.

Therefore, a question worthy of serious consideration is: Is there a common molecular event that precedes the occurrence of insulin resistance and SASP in time, and connects mitochondrial metabolism with epigenetic regulation in space?

II. How a High-Calorie Diet Triggers a Metabolic Crisis: From "Reductive Stress" to ROS Burst

2.1 Reductive Stress: A Long-Ignored Metabolic Imbalance Traditional theory attributes the pathogenesis of metabolic diseases to "oxidative stress" (excess ROS), but recent studies suggest that reductive stress—the excessive accumulation of reducing equivalents such as NADH and NADPH—may be an equally critical, or even more upstream, driving factor. A 2025 review published in Free Radical Biology and Medicine systematically elaborated this mechanism: under conditions of high calorie, high carbohydrate, and high fat intake, a large amount of electron donors floods into mitochondria, causing a "traffic jam" in the electron transport chain—NADH and NADPH concentrations rise abnormally, electrons accumulate at complexes I and III, leading to partial inhibition or reverse electron flow.

"Abnormally high concentrations of NADH and NADPH create an electron 'traffic jam' in the mitochondrial electron transport chain, causing partial inhibition of upstream complexes or reverse electron flow. This highly reduced environment promotes ROS generation by increasing electron leakage to molecular oxygen."

2.2 The Double Blow of High Carbohydrate and High Fat A high-carbohydrate diet causes a surge in glycolytic flux, with large amounts of pyruvate entering mitochondria, producing excess acetyl-CoA and NADH. At the same time, a high-fat diet (especially diets rich in polyunsaturated ω-6 fatty acids) directly infuses large amounts of electron donors into the electron transport chain.

The combined result is: the rate of NADH production far exceeds the processing capacity of the electron transport chain, and the NADH/NAD⁺ ratio continues to rise. This state is called "reductive stress." Its essence is that the cell, due to "fuel excess," falls into metabolic congestion—not a lack of energy, but too much energy, causing the electron transport chain to "jam," which in turn leads to decreased ATP synthesis efficiency and an explosive increase in ROS.

Multiple studies have confirmed that a high-calorie diet downregulates mitochondrial oxidative phosphorylation (OXPHOS), while antioxidant and stress response defense systems are also simultaneously suppressed. This means that not only is there a problem on the "production side" of ROS, but the "clearance side" is also failing in sync.

III. Sensitivity of Key Metabolic Enzymes to ROS: Why Is Aconitase the "First Target"?

3.1 From Glycolysis to the TCA Cycle: Key Enzymes in Multiple Metabolic Pathways Are Inhibited by ROS In the complete oxidation process from glucose to carbon dioxide and water, multiple key metabolic enzymes are highly sensitive to ROS:

3.2 Aconitase: The "First Target" of ROS Attack Among all the above enzymes, aconitase, due to its unique [4Fe-4S] cluster structure, becomes the "preferred target" of ROS attack. A 2019 review published in Accounts of Chemical Research clearly pointed out: aconitase is a major target of reactive oxygen and nitrogen species (superoxide anion O₂•⁻, hydrogen peroxide H₂O₂, nitric oxide •NO, peroxynitrite ONOO⁻). These reactive intermediates can oxidize the iron-sulfur cluster, leading to iron release and loss of aconitase catalytic activity.

"Accumulated experimental evidence indicates that aconitase is a major target of reactive oxygen and nitrogen species. These intermediates can oxidize the iron-sulfur cluster, leading to iron release and loss of aconitase catalytic activity."

Structural basis for aconitase becoming the "first target":

1. Solvent      exposure of the Feₐ site: In the [4Fe-4S] cluster      of aconitase, only three iron ions are coordinated with cysteine thiol      ligands, while the fourth iron ion (Feₐ) is directly exposed to the solvent and binds to      the substrate citrate. This structure makes Feₐ extremely vulnerable to ROS attack.

2. Reaction rate      with superoxide anion: The reaction      rate between the aconitase Fe-S cluster and superoxide anion is as high as      10⁷ M⁻¹s⁻¹. This rate is faster than the rate at which most      antioxidant enzymes clear superoxide anion, meaning that at the moment ROS      are generated, aconitase is immediately hit.

3. Order-of-magnitude      difference in inactivation rate: A 2023 study published in Free Radical Biology and Medicine      directly measured the reaction rate constants of aconitase with various      ROS: the reaction rates with superoxide anion and carbonate radical are      approximately 10⁸ M⁻¹s⁻¹, with peroxynitrite      approximately 10⁵ M⁻¹s⁻¹, and with hydrogen      peroxide approximately 10² M⁻¹s⁻¹. These rate constants      indicate that aconitase's sensitivity to ROS is at the highest level among      metabolic enzymes.

4. Irreversible      damage after oxidation: The      [4Fe-4S]²⁺ cluster of aconitase, after      oxidation, forms an inactive [3Fe-4S]⁺ cluster, leading to loss of enzyme activity. A      2014 metabolic control analysis study published in Free Radical      Research provided key quantitative evidence: the iron-sulfur      cluster of aconitase can be rapidly and selectively inactivated by      oxidants. Under equal ROS exposure conditions, the inactivation rate      of aconitase is far higher than that of other iron-sulfur      cluster-containing enzymes. The study also found that the flux control      coefficient of aconitase over respiration exceeds 0.95 in the brain and      heart, meaning that in these high-energy-consuming tissues, a slight      decrease in aconitase activity can significantly affect mitochondrial      respiration and ROS production.

3.3 Risk of Self-Amplification More critically, the Fe²⁺ released after Feₐ dissociation from the iron-sulfur cluster can undergo the Fenton reaction with hydrogen peroxide, generating hydroxyl radicals (·OH)—a more destructive oxidizing species than the initial ROS. This means that once aconitase is inactivated, it itself becomes an "igniter" for further ROS diffusion, rather than a mere victim.

In addition, the 2023 study also found that the thermally stability of the oxidized [3Fe-4S]⁺ state is significantly reduced, with the melting temperature dropping from 51.1°C to 43.6°C, further exacerbating its structural collapse under oxidative stress.

Therefore, the inactivation of aconitase is not only a damage event but also an "accelerator" of metabolic collapse—it causes TCA cycle congestion, electron transport chain congestion, and ROS production to form a self-accelerating positive feedback loop.

IV. Citrate Efflux: The Common Starting Point of Two Pathological Branches

4.1 SLC25A1-Mediated Citrate Efflux When aconitase activity decreases, citrate cannot be smoothly converted to isocitrate, and its concentration in the mitochondrial matrix increases. When this accumulation reaches a certain threshold, it drives enhanced efflux activity of SLC25A1 (mitochondrial citrate transporter, also known as tricarboxylate carrier, CIC). Studies have shown that the transport of mitochondrial citrate to the cytoplasm by SLC25A1 plays important regulatory roles in glycolysis, lipid synthesis, and protein acetylation. ACLY (ATP-citrate lyase) then cleaves citrate in the cytoplasm into acetyl-CoA and oxaloacetate. A 2020 study published in Nature Communications directly confirmed that nitric oxide (NO) reprograms macrophage metabolism by targeting aconitase 2 (ACO2) and pyruvate dehydrogenase (PDH). Using ¹³C tracing and mitochondrial respiration experiments, the study precisely localized NO-mediated metabolic inhibition to mitochondrial aconitase—this is direct evidence of aconitase as a "metabolic reprogramming switch."

4.2 Branch One: Metabolic Stress Pathway (→ Insulin Resistance) Acetyl-CoA generated from citrate in the cytoplasm generates malonyl-CoA through the de novo fatty acid synthesis pathway, and then synthesizes diacylglycerol (DAG). DAG activates protein kinase Cθ (PKCθ), which phosphorylates serine residues of insulin receptor substrate 1/2 (IRS-1/2). This phosphorylation event prevents normal tyrosine phosphorylation of IRS-1/2, thereby blocking insulin receptor downstream signaling. The result is: GLUT4 vesicles cannot translocate to the cell membrane, and insulin-stimulated glucose uptake is reduced—this is precisely the core mechanism of lipid-induced insulin resistance.

Disease spectrum involved: type 2 diabetes, non-alcoholic fatty liver disease, PCOS, hypertriglyceridemia.

4.3 Branch Two: Inflammaging Pathway (→ SASP) A study published on July 29, 2026, in Nature (IF 56.1) revealed a novel SASP regulatory mechanism independent of cGAS-STING. The study found: in senescent cells, the mitochondrial pyruvate-citrate-acetyl-CoA axis is upregulated, increasing acetyl-CoA availability to support histone acetylation on SASP genes. As a substrate for histone acetyltransferases (HATs), acetyl-CoA increases H3K27ac modification in the promoter regions of SASP genes, promoting chromatin opening and pro-inflammatory transcription.

The study further confirmed that inhibiting SLC25A1 (mitochondrial citrate transporter) reduces histone acetylation at SASP loci, limiting this inflammatory program. In in vivo experiments, inhibiting SLC25A1 also reduced chromatin accessibility at SASP loci, suppressed inflammation, and improved healthspan in aged mice.

In addition, the study found that LPS (lipopolysaccharide) and cytokines such as TNFα and IFNγ can activate SLC25A1 and ACLY genes in immune cells, and CIC and ACLY activities are required for macrophage inflammatory responses. This further confirms the core position of the citrate-acetyl-CoA pathway in inflammatory regulation.

Disease spectrum involved: arteriosclerosis, Alzheimer's disease, osteoarthritis, immune aging.

4.4 Positive Feedback Synergy of the Two Branches The two branches do not operate in parallel without interaction; rather, they form positive feedback reinforcement through the following mechanisms:

• Branch one      (insulin resistance) leads to      hyperglycemia, which increases ROS through glucose autoxidation and the      polyol pathway, further exacerbating persistent oxidative damage to      aconitase;

• Branch two      (SASP) secretes      pro-inflammatory factors such as TNF-α and IL-6, which activate the NF-κB      pathway, on the one hand upregulating SLC25A1 and ACLY expression, and on      the other hand inhibiting mitochondrial Complex I and aconitase      expression.

Thus, a self-accelerating metabolic collapse loop centered on aconitase is formed.

It should be emphasized that this is still a mechanistic hypothesis, particularly regarding the direct causal relationship between aconitase inactivation and enhanced SLC25A1 efflux, which still requires more experimental validation. However, this hypothesis has clear testability and falsifiability.

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