Nuclear Factor Erythroid 2 Related Factor 2 and Mitochondria Form a Mutually Regulating Circuit in the Prevention and Treatment of Metabolic Syndrome

Oxidative stress and inflammation serve as central risk factors of MetS

Oxidative stress and inflammation are both the major causes and symptoms of MetS, as summarized in Figure 1. Oxidative stress is mainly defined as the imbalance in production and degradation of ROS, which is closely related to the occurrence of a variety of metabolic disorders. Energy disturbance and redox imbalance are the roots of a variety of metabolic disorders, collectively known as MetS (Chen et al., 2017). MetS covers a range of risk factors, mainly including abnormal blood glucose, elevated blood pressure and triglyceride levels, reduced HDL-C levels, insulin resistance, and obesity (Alberti et al., 1998). These risk factors could trigger a surge in ROS release in numerous cell types or tissues (including vascular endothelial cells, myocytes, or pancreatic beta-cells).

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FIG. 1.

Amelioration of oxidative stress and inflammation in MetS through the actions of Nrf2 and mitochondria. Oxidative stress and inflammation are both the major causes and symptoms of MetS. By targeting oxidative stress and inflammation, Nrf2 and mitochondria may form a mutually regulated circuit to effectively prevent and improve MetS, such as obesity, diabetes, and cardiovascular disease. (A) Possible mechanisms of Nrf2 in the regulation of MetS. (B) Possible mechanisms of mitochondria in the regulation of MetS. FFA, free fatty acid; IL-6, interleukin-6; MetS, metabolic syndrome; Nrf2, nuclear factor erythroid 2 related factor 2; TNF-α, tumor necrosis factor-α. Color images are available online.

A variety of studies suggest that treatments that reduce ROS overproduction may also improve insulin sensitivity, hyperlipidemia, hepatic steatosis, and vascular endothelial disease (Wang et al., 2016). High-sugar and high-fat diets (HFD) are frequently accompanied by a dramatic rise in ROS release in vivo, whereas inhibition of the NOX1/MCP1 cascade in differentiated adipocytes contributes to the reduction of ROS release, thereby delaying the onset of MetS such as obesity and insulin resistance (Den Hartigh et al., 2017; Han et al., 2012). Improving hyperlipidemia through the Wnt/β-catenin pathway, ameliorating NAFLD by inhibiting toll-like receptor 4 (TLR4)/NOX2 activation, and repairing vascular endothelial disease by reducing excessive autophagy all essentially reduce ROS release (Chen et al., 2019; Kim et al., 2017; Wang et al., 2021).

In addition, ROS may modulate a variety of signaling pathways, including mitogen-activated protein kinase (MAPK), p53, PI3K/AKT, etc. Therefore, antioxidants may improve MetS by scavenging excess ROS and thereby modulating these signaling pathways (Satoh et al., 2021). For instance, a compound called celastrol binds to CAP1 and inhibits the interaction between CAP1 and resistin, thus inhibiting the protein kinase A (PKA)/nuclear factor kappa-B (NF-κB) signaling pathway and excessive ROS release to ameliorate HFD-induced MetS in mice (Zhu et al., 2021). Similarly, N-acetylcysteine maintains mitochondrial redox homeostasis by activating the SIRT3/SOD2/Gpx4 signaling pathway and reduces ROS levels to attenuate MetS (Li et al., 2022a).

Furthermore, the imbalance between ROS clearance and production further promotes oxidative stress and inflammation (Castro et al., 2016). Lipid peroxidation, protein carbonylation, increase of NOX activity and decrease of glutathione (GSH) level in MetS may lead to enhanced ROS formation (Holvoet et al., 2004). ROS proliferation could further promote the aggravated symptoms of obesity, diabetes and CVD by decreased antioxidant response and altered gene expressions of inflammatory cytokines, chemokines and growth factors.

Inflammation is another major cause of MetS. In addition to ROS, free fatty acid (FFA) induced by HFD or obesity in MetS is the crux factor of inflammation (Xu et al., 2022). Two stages of inflammation exist: acute and chronic. Acute inflammation is usually the non-specific response. When acute inflammation lasts for a longer period of time, the second stage of inflammation (chronic inflammation) occurs.

In the development of obesity, enlargement of adipocytes occurs, accompanied by the release of FFA and inflammatory cytokines in large amounts from the adipose tissue, resulting in not only hyperlipidemia but also systemic lipid accumulation and inflammation. The common feature of diabetes is elevated blood glucose level, also known as hyperglycemia, which occurs mainly due to either insufficient insulin secretion or insulin resistance.

FFA plays a key role in both insulin resistance and hyperglycemia. FFA serves as a catalyst to augment glucose production by stimulating PKA, which is subsequently enlisted by cAMP response element binding (CREB) to the regulatory domain of genomic gluconeogenesis, thereby fostering hepatic gluconeogenesis (Zhao et al., 2023), and FFA instigates insulin resistance via several pathways, primarily by initiating the TLR4/tumor necrosis factor-α (TNF-α) cascade inflammatory response (Liu et al., 2019; Sears and Perry, 2015).

Furthermore, when the distribution of glucose in glycogen is reduced, FFA will promote elevated triglycerides, and hypertriglyceridemia may lead to microangiopathy in the kidneys and trigger the pancreas to secrete a large amount of insulin (Sieber and Jehle, 2014), which in turn produces hyperinsulinemia, resulting in increased glucose uptake by the kidneys to cause side effects on the kidneys (Guo et al., 2018). Of note, excessive adipose tissue also generates large amounts of FFA in blood vessels, which may promote vascular endothelial dysfunction (Liu et al., 2019). Endothelial dysfunction is an early symptom of atherosclerosis and acute coronary syndrome (Shao et al., 2020). When endothelial dysfunction occurs, vascular resistance increases and the secretion of angiotensin II, catecholamine, and aldosterone, which easily stimulate cardiomyocyte hypertrophy, and aggravate myocardial hypoxia and ischemia (Stier et al., 2005), or trigger acute myocardial infarction because of the viscosity of the blood (Nakada et al., 2017).

Moreover, hyperinsulinemia in diabetes may enhance sodium reabsorption, increase sympathetic nervous system activity, and elevate circulating FFA levels, therefore promoting CVD such as hypertension (Davignon and Ganz, 2004). Concurrently, insulin resistance results in the excessive production of ROS and proinflammatory cytokines (interleukin [IL]-6, TNF-α), and elevated levels of ICAM-1 and VCAM-1 (Victor et al., 2016), which may contribute to the formation of endothelial dysfunction, myocardial hypertrophy, or other CVDs (Borthwick et al., 2012). Thus, inflammation in MetS occurrence forms a circulatory conduction from adipose tissue to liver, kidney, blood vessels, and heart through FFA.

In MetS, Nrf2 mediates lipid synthesis and fatty acid oxidation through regulating its downstream genes, such as RXRa/peroxisome proliferators-activated receptors γ (PPARγ), thus directly or indirectly manipulates mitochondrial lipid transport in adipose and liver tissues (Samaha et al., 2022; Zhao et al., 2020). In addition, Nrf2 regulates pancreatic lipid metabolism through the Pparγ/p62-mediated “antioxidant–autophagy” axis (Liu et al., 2022). More details are described in Mitochondria and Nrf2 Form a Mutually Regulating Circuit to Improve MetS section.

Nrf2 targets oxidative stress and inflammation

Nrf2 is a member of the Cap‘n'collar transcription factor family (mitochondrial transcription factor A [Tfam]), consists of 605 amino acids, and is divided into 7 highly conserved basic leucine zipper structures, known as Nrf ECH homology 1–7 (Neh1–7) (Fig. 2A). Keap1 is a cullin3 (Cul3) ringbox 1 (Rbx1) adaptor protein containing E3 ubiquitin ligase (Fig. 2B) (Katoh et al., 2005), which facilitates Nrf2 ubiquitination and its proteasomal degradation under normal physiological conditions (Zhang and Hannink, 2003). When cells undergo attack by electrophilic substances or ROS, Nrf2 is released from Keap1–Cul3–Rbx1 complex and translocated into the nucleus, wherein it heterodimerizes with small Maf proteins (Ma et al., 2019; Rojo et al., 2012). After releasing, Nrf2 activates its downstream genes transcription with the help of Neh4, Neh5, and CREB protein by targeting antioxidant response element (ARE) in the gene regulatory region (Fig. 2C) (Rushmore et al., 1991).

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FIG. 2.

Intracellular regulation of Nrf2 and Keap1. Nrf2 is an essential transcription factor in regulating cellular oxidative stress response, mitigating ROS and electrophiles-induced cellular damage by regulating the production of a range of antioxidant proteins. (A) In human Nrf2 protein, there are seven highly conserved bZIP structures, Neh1–7. Neh1 contains CNC bZIP region, and could form a dimer with sMaf and bind to DNA; Neh2 domain can negatively accommodate Nrf2 through DLG and ETGE; Neh3 at C-terminal is an active region, which can aggregate CHD6 protein; Neh4 and Neh5 mainly aggregate CREB-binding protein RAC3; Neh6 domain can negatively manage Nrf2 β-TrCP dimer through DSGIS and DSAPGS sequences; Neh7 inhibits Nrf2 by mediating the physical interaction between RXR and ARE. (B) Keap1 is a Cul3 Rbx1 adaptor protein containing E3 ubiquitin ligase. It can bind to the N-terminal of Cul3 through IVR domain, and then cooperate with Cul3-Roc1 to promote Nrf2 degradation by 26S proteasome. (C) At the onset of stress, Nrf2 releases from Keap1 and enters into nucleus to bind with ARE, and (D) further promotes a series of downstream antioxidant gene expressions. (E) Nrf2-mediated pathway could promote mitochondrial function, and mitochondrial-generated ROS could also inversely regulate Nrf2-related gene expressions. ARE, antioxidant response element; bZIP, basic leucine zipper; CHD6, chromo-ATPase/helicase DNA binding; CNC, cap‘n'collar; CREB, cAMP response element-binding protein; Cul3, cullin3; DLG, Asp-Leu-Gly; ETGE, Glu-Thr-Gly-Glu; Keap1, Kelch-like ECH-associated protein 1; Neh1–7, Nrf ECH homology 1–7; RAC3, receptor-associated coactivator 3; Rbx1, ringbox 1; ROS, reactive oxygen species; RXR, retinoid X receptor; β-TrCP, β-transducin repeat containing protein. Color images are available online.

As a “master regulator” of antioxidant response (Zhang et al., 2018), Nrf2 modulates the expression of hundreds of genes or antioxidant enzymes. Its common downstream genes include NADPH quinone oxidoreductase 1 (NQO1), glutathione sulfotransferases, glutamate cysteine ligases, heme oxygenase 1 (HO-1), acetaldehyde dehydrogenase-1/2, GSH peroxidase (Gpx), glutathione reductase, γ-glutamylcysteine synthetase (γGCS), thioredoxin reductase (TrxR) or thiourea reductase, glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), isocitrate dehydrogenase 1, malic enzyme 1, SOD, etc. (Fig. 2D) (Wu et al., 2011).

Notably, recent studies revealed that more downstream genes of Nrf2, such as metallothionein, play a prominently protective role in MetS-related diseases such as diabetic nephropathy (Wu et al., 2016), diabetic cardiomyopathy (Gu et al., 2017), and liver injury (Yu et al., 2021). Nrf2 could improve inflammation by regulating cell cycle-related protein phosphorylation and ubiquitination, cell growth and apoptosis-related protein transport, such as preventing IκBα degradation by mediating HO-1 (Yang et al., 2013), or inhibiting proinflammatory cytokines (IL-1, IL-6, TNF-α, COX2) through NF-κB interaction with macrophages (Saha et al., 2020).

Nrf2 may also improve MetS by altering metabolic reprogramming during cellular oxidative stress or inflammation in diverse ways, involving suppressing adipogenesis, supporting fatty acid β-oxidation, promoting the pentose phosphate pathway, balancing GSH levels, and enhancing NADPH regeneration and purine synthesis. Moreover, the ubiquitin–proteasome system plays an essential role in protein quality control and homeostasis in cells (Fig. 1).

Proteasome activity and autophagy are controlled by Nrf2, which removes misfolded proteins, chronically accumulated protein aggregates, and damaged mitochondria (Park et al., 2020) (Fig. 2E), peroxisomes and ribosomal proteins (Levine et al., 2011). So, Nrf2-Keap1 is implicated in regulating the rate-limiting steps of metabolism or regulating enzymes located at major branch points in metabolic pathways, thereby assisting the synthesis of carbohydrates, nucleic acids, lipids, and amino acids to improve MetS.

Nrf2 activators

Based on the mechanism of Nrf2 in the regulation of MetS, we have classified a group of compounds that activate Nrf2 directly or indirectly as Nrf2 activators. These compounds could improve MetS by enhancing the antioxidant and anti-inflammatory defense systems. As shown in Table 1, we list some representative pathways of Nrf2 activators. Generally, Nrf2 activators activate Nrf2 via Keap1 electrophilic cysteine modification, GSK3β/β-Trcp inhibition, p62/autophagy upregulation, protein–protein interaction (PPI) interruption, or some other alternative mechanisms, as shown in Figure 3.

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FIG. 3.

Summary of the pharmacological strategies to modulate Nrf2 activity. The majority of Nrf2 activators are Keap1 inhibitors, Keap1-independent inhibitors, and protein–protein interrupters (PPI). Keap1 inhibitors covalently modify cysteine residues in the thiol-rich KEAP1 protein by oxidation or alkylation, producing a closed state; or by interaction with the Cul3/Rbx1 complex. Inhibitors of Keap1-independent modalities may also activate Nrf2. P62 negatively regulates the Nrf2 pathway by removing Keap1 and enhancing the abundance of Nrf2. Hrd1 is localized in the ER and is able to react with Xbp1 protein. In addition, GSK3β and β-TrCP act as alternative mechanisms to Keap1 to inhibit the activation of Nrf2. GSK3β acts on Fyn to bring it into the nucleus, where Fyn phosphorylation leads to Nrf2 and nuclear export. Activation of the PI3K/Akt pathway also increases the abundance of Nrf2. PPI inhibitors interfere with the docking of Nrf2 with the Kelch propeller of Keap1 to activate Nrf2. Akt, protein kinase B; ER, endoplasmic reticulum; Fyn, a tyrosine protein kinase; Hrd1, a HMG-CoA reductase; PPI, protein–protein interaction; Xbp1, X-box–binding protein 1. Color images are available online.

The dark side of Nrf2

Nrf2-mediated myocardial injury was first identified in 2013 in aging cardiomyocyte-restricted human mutant CryAB transgenic mice (Kannan et al., 2013). Later, Qin et al. (2016) reported that Nrf2 could mediate cardiac maladaptive remodeling induced by pressure overload. Impaired cardiac autophagy may occur in the event of myocardial hypertrophy or dysfunction. In such cases, Nrf2 activation can be detrimental to the heart, predisposing it to pathological activation of the renin–angiotensin system, and adversely affecting cardiac remodeling and function (Zang et al., 2020). There are also some dark sides of Nrf2 in insulin resistance.

Emerging evidence suggests that lack of Nrf2 improves insulin resistance, adipogenesis, and adipocyte differentiation. Overexpression of Nrf2 gene also induces insulin resistance under certain conditions, which has been reviewed by Zhang et al. (2015). Studies on skeletal muscle suggest that excessively prolonged Nrf2 activation may induce insulin resistance by impairing insulin signaling and glucose uptake (Xu et al., 2012), as well as decreasing ROS levels (Schottlender et al., 2021).

Another challenge of Nrf2 activation in the treatment of MetS is the cytotoxicity caused by off-target effects. The crystal complex structure of Neh2 domain of Nrf2 may interfere with the binding of Keap1 to Nrf2, resulting in off-target effect. Researchers have withdrawn CDDO-methyl ester from a phase III clinical trial in patients with end-stage T2D kidney disease because of its off-target effects, although no serious adverse events or deaths were reported (Chin et al., 2018). Topical treatment of CVD can transport the activated Nrf2 complex to the designated site to avoid potential off-target toxicity (Keap1 and Nrf2 sulfhydryl reaction may have off-target toxicity) (Rojo et al., 2012).

In addition, due to the different bioavailability and action targets, Nrf2 activators have great differences in efficacy. These could be changed according to the different ways of taking and formula. Therefore, more work needs to be done to improve the stability of Nrf2 activators in applications. Notably, the circuit formed by Nrf2 and mitochondria enables a more stable efficacy of Nrf2 activators and also reduces their side effects.

Nrf2 and mitochondrial biogenesis

Nrf2 may be associated with the regulation of mitochondrial biogenesis. Activation of Nrf2 could enhance the expression of nuclear respiratory factor 1/2 (NRF1/2) and Tfam, which are key regulators in mitochondrial biogenesis (Hock and Kralli, 2009; Lin et al., 2023). The NRFs NRF1 and NRF2 act on most of the nuclear genes that specify the subunits of the five respiratory complexes of the mitochondrial inner membrane, in addition to participating in regulating the expression of mitochondrial transcription factors. Therefore, NRF1 and NRF2 are closely related to nuclear–mitochondrial interaction (Scarpulla, 2008).

Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), another key regulator in maintaining mitochondrial mass, is reported to promote mitochondrial biogenesis by crosstalk with Nrf2 (Liu et al., 2009). A previous study has shown that endogenous carbon monoxide generated by HO-1 overexpression in cardiomyocytes stimulates mitochondrial H₂O₂ production and activates AKT to induce Nrf2 nuclear translocation, followed by upregulation of NRF1/α-PAL encoding nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 1 and COX1/complex IV to promote mitochondrial biogenesis (Esteras et al., 2023; Piantadosi et al., 2008).

On the contrary, the mitochondrial biogenesis-related protein PGC-1α could inversely regulate Nrf2. PGC-1α stabilizes Nrf2 by inactivating GSK3β through a p38-dependent pathway (Choi et al., 2017). Thus, Nrf2 helps maintain normal mitochondrial biogenesis and intracellular redox homeostasis by interacting with mitochondrial PGC-1α (Fig. 4A).

Furthermore, as a well-known regulator of mitochondrial energy metabolism, AMPK activation could improve mitochondrial biogenesis via activating PGC-1α (Cao et al., 2015). Our previous study showed that AMPK/Akt may act as an upstream signaling pathway of both PGC-1α and Nrf2 to simultaneously regulate mitochondrial biogenesis and intracellular antioxidant system, which could be another regulatory relationship between PGC-1α and Nrf2 (Fig. 4B).

Nrf2 and mitochondrial redox

Mitochondria are the main organelles to regulate intracellular redox, and the critical enzymes in this process are controlled by Nrf2. First, within mitochondria, Gpx1/Gpx4 uses GSH and NADPH to detoxify superoxide-derived H₂O₂ into water to maintain mitochondrial redox homeostasis (Zou et al., 2014). In this process, Nrf2 activation can upregulate Gpx1/Gpx4 expression, and increase GSH and NADPH levels. Second, Nrf2 is involved in the regulation of expressions of mitochondrial antioxidant enzymes, including SOD2, Prdx3, Prdx5, GPx1, and TrxR2 (Cao et al., 2014; Kasai et al., 2020; Zhu et al., 2010). In addition, NOX2 and NOX4 are closely associated with ROS release, both of which could be modulated by Nrf2 (Kovac et al., 2015; Sun et al., 2017).

SIRTs, a member of the NAD⁺-dependent deacetylase family, are involved in the regulation of Ca²⁺ homeostasis, mitochondrial lipid metabolism, and cellular senescence in vivo and in vitro (Zia et al., 2021). SIRT1, as the most widely studied SIRT in mammals, promotes the transcriptional activity of Nrf2 through deacetylation of Nrf2 (Ding et al., 2016). In a rat model of cerebral ischemia–reperfusion injury, SIRT1 directly activates the Nrf2/HO-1 pathway and regulates mitochondrial reactive oxygen species (mtROS) release (Valero, 2014; Zeng et al., 2023).

Meanwhile, SIRT1 may also deacetylate LKB1, the upstream of AMPK to activate AMPK, cascade with PGC-1α to regulate mitochondrial biogenesis (Shi et al., 2020), and ameliorate mitochondrial injury in a SIRT1/PGC-1α/eIF2α/ATF4 cascade manner (Hayakawa et al., 2015). Similarly, SIRT3 acts as a deacetylase to deacetylate and activate Nrf2, initiating a series of antioxidant genes (Fig. 4C) (Kim et al., 2022; Wang et al., 2019b).

Conversely, Nrf2 activation is tightly linked to mitochondrial redox status. The generation of ROS by mitochondria may occur due to electron leakage in the electron transport chain (ETC). Metabolic stress and chemical agents such as paraquat can cause a remarkable increase in mtROS generation by activating NOX or inhibiting SOD activity, further activating the Nrf2-mediated antioxidant pathway to combat oxidative stress (Fig. 4D) (Cox et al., 2018; Robb et al., 2015). Eukaryotes have three types of SOD: SOD1 encodes cytoplasmic Cu/Zn-SOD; SOD2 encodes mitochondrial Mn-SOD, and SOD3 encodes extracellular SOD (Gao et al., 2020).

SOD2 converts superoxide to O₂ ⁻ and H₂O₂, which can be further converted to water by Prdx3/5 (Zhu et al., 2010). It has been reported that ROS release from mitochondria in response to acute exercise stress activates the Nrf2/ARE signaling pathway to enhance anti-inflammatory response in the mouse heart. In this process, mtROS activates the Nrf2 pathway mainly through NOX4 and SOD2 interactions, but not NOX2 and SOD1 (Casciaro et al., 2018).

Myocardium-specific NOX4-KD will significantly affect mitochondrial mass, leading to metabolic stress, decreased ATP levels, and increased NAD⁺/NADH ratio. ATF4, a key protein responsible for poststress effects in mitochondria, may activate Nrf2 to maintain intracellular redox homeostasis via upregulating GSH and NADPH (Kasai et al., 2020).

Hence, Nrf2 could be activated in response to ROS generated by mitochondria or NADPH oxidases and also by some mitochondria-related antioxidant enzymes such as Prdxs, NOXs, ATF4, SIRT1, and SODs.

Nrf2 and mitochondrial dynamics

Mitochondria dynamics mainly consist of mitochondrial fission and fusion. Mitochondrial fusion promotes mitochondrial extension, and mitochondrial fission increases mitochondrial breakage. Mitofusin-1/2 (MFN1/2) and optic atrophy 1 (OPA1) mediate fusion, and dynamin-1 like protein (DRP1) promotes fission (Chan, 2012). Impaired mitochondrial dynamics are manifested by abnormal or broken mitochondrial structure, impaired turnover, abnormal ROS release, and mtDNA mutations, resulting in changes in mitochondria-derived signaling and abnormal energy metabolism. It has been reported that diabetic patients with reduced Nrf2 level in endothelial progenitor cells showed dysregulated abundance of mitochondrial dynamics proteins DRP1, OPA1, or MFN1/2, leading to mitochondrial disruption and endothelial progenitor cell dysfunction, while overexpression of Nrf2 attenuated mitochondrial disruption or dysfunction, and recovered mitochondrial dynamics protein abundance (Dai et al., 2022).

Also, in lead-induced neurocytotoxic injury, the AMPK/Nrf2 pathway can be activated to improve mitochondrial fission, and promote energy supply and cell survival (Yang et al., 2020). Multiple Nrf2 transcription factors have been reported to be required for a hyperfused mitochondrial network caused by Keap1 knockdown. Furthermore, increased Nrf2 activity results in elevated proteasome activity to degrade the mitochondrial fission protein DRP1 (Sabouny et al., 2017).

Conversely, changes in these mitochondrial fusion/fission-related proteins may also alter Nrf2 abundance. Interestingly, OPA1 or MFN1/2 KO impairs mitochondrial network structure, which drives ROS-triggered Nrf2 retrograde signaling to inhibit self-renewal of neural stem cells (Khacho et al., 2016). A similar scenario occurs in breast cancer cells cultured on a soft cell matrix under conditions, whereby increased mitochondrial f-actin is accompanied by increased DRP1 and mief1/2-dependent mitochondrial fission, leading to mtROS production that reversely activates Nrf2 transcription (Fig. 4E) (Romani et al., 2022).

Nrf2 and mitochondrial energy metabolism

Nrf2 is necessary for maintaining normal mitochondrial OXPHOS and energy metabolism. In the absence of Nrf2, the impaired activity of mitochondrial complex I due to substrate limitation caused by the reverse electron flow of complex II is one of the major causes of ROS overproduction. Nrf2-KD cells have significantly lower rates of NADH and FADH2 production, resulting in limited electron supply to the mitochondrial ETC. Mitochondrial membrane potential (ΔΨm), oxygen consumption rate, and ATP production levels were lower in Nrf2-deficient mouse embryonic fibroblasts (Ryoo and Kwak, 2018). Nrf2 also plays a decisive role in the ATP production pathway.

In Nrf2-KO cells, glycolytic intermediates (pyruvate and lactate) and products of glycolysis (G6PD, 6PGD, dihydroxyacetone phosphate) are closely related to mitochondrial energy metabolism, indicating that the main pathway of ATP production changes from OXPHOS to glycolysis (Mitsuishi et al., 2012). Notably, in the absence of Nrf2, the ATP produced by glycolysis is partially used by the F1F0 ATPase to maintain the ΔΨm (Fig. 4F) (Moser et al., 2009).

In turn, alteration in mitochondrial OXPHOS function may also affect Nrf2 expression and activation. In cancer cells, increased mitochondrial complex I activity stimulates ERK5-dependent myocyte enhancer factor 2 activation and induces Nrf2 expression in a ROS-dependent manner. Of note, Nrf2 activity is usually reduced after impaired mitochondrial OXPHOS function, which is mediated by the decline of complex I, ERK5, and MEF2 in a ROS-independent way (Khan et al., 2018). Interestingly, the amount of succinate, an intermediate of the tricarboxylic acid (TCA) cycle, is also involved in the Nrf2–mitochondria axis during ATP production by mitochondrial OXPHOS.

As a mitochondrial metabolite, the succinate concentration is dramatically elevated when mitochondrial energy metabolism is impaired. When cells undergo oxidative stress, succinate binds specifically to succinate receptor 1 (SUCNR1), contributing to the release of ROS via the mitochondrial complex II, and also facilitates inflammation through macrophage activation (Milliken et al., 2022). High levels of SUCNR1 may stimulate Nrf2 to activate a series of antioxidant genes to protect mitochondria from oxidative damage (Holmstrom et al., 2013).

Succinate reduction and SUCNR1 signaling may profoundly affect inflammatory and immune responses. In contrast, itaconic acid, as a metabolite of the TCA cycle, is a powerful anti-inflammatory metabolite that activates Nrf2 and ATF3 responses during anti-inflammatory processes.

Nrf2 and mitochondrial glycolipid metabolism

Mitochondria-related glycolipid metabolism is also affected by Nrf2. As mentioned above, glycolysis intermediates pyruvate and lactate were significantly elevated in various Nrf2-KD cancer cells (Mitsuishi et al., 2012). Furthermore, mitochondria are involved in glycolipid metabolic pathways such as the TCA cycle and fatty acid β-oxidation (Liu et al., 2021). The key rate-limiting steps in the pentose phosphate pathway include G6PD-catalyzed conversion of glucose-6-phosphate to 6-phosphogluconate and 6PGD-catalyzed further conversion of 6-phosphogluconate to ribose 5-phosphate, both of which could be modulated by Nrf2 (Sun et al., 2015; Zhao et al., 2015).

In the case of glucose deprivation, the OXPHOS responses of Nrf2-KO cells rely mainly on mitochondrial fatty acid β-oxidation to provide substrates. Moreover, under Nrf2-deficient conditions, fatty acid β-oxidation is inhibited and ATP synthesis is lower. In Nrf2-KO 293T cells, carnitine palmitoyltransferase (CPT) expression is reduced (Pang et al., 2014; Sharma et al., 2018). Moreover, reduced AMPK phosphorylation level was observed in the liver of Nrf2-KO mice (Meakin et al., 2014), which inhibits downstream acetyl-CoA carboxylase (ACC) (Ser79) phosphorylation, leading to increased ACC and fatty acid synthase expression (Fig. 4G) (Hou et al., 2016).

In addition, Nrf2 directly modulates gene expression of malic enzyme 1, a crucial enzyme in the TCA cycle, to limit the supply of pyruvate (Thimmulappa et al., 2002). Interestingly, during diabetic kidney disease, activation of Nrf2 not only directly activates the transcription of the gene encoding the glycolytic enzyme but also inhibits the conversion of pyruvate to acetyl coenzyme A by directly activating pyruvate dehydrogenase kinase 1, leading to inhibition of the TCA cycle, thereby promoting the “Warburg's Effect” (Chang et al., 2018; Sas et al., 2016; Sharma et al., 2013).

Consequently, Nrf2 impacts mitochondrial glycolipid metabolism by regulating key rate-limiting enzymes and generating substrates in gluconeogenesis, pentose phosphate pathway, fatty acid β-oxidation, and TCA cycle.

Nrf2 and autophagy

Nrf2 may also affect mitochondrial homeostasis by regulating autophagy, especially mitophagy. Nrf2-KO mice showed an overall decrease in autophagic turnover, including dramatic accumulation of p62 and attenuation of LC3-II, suggesting that autophagy is dependent on Nrf2 activation (Chang et al., 2015). Nrf2 is also involved in mitophagy, a process that involves selective elimination of excess or damaged mitochondria before degradation by lysosomes. In the absence of Nrf2, Keap1 begins to degrade Miro2, a mitochondrial outer membrane protein, which can serve as a Parkin receptor for selective removal of damaged mitochondria (O'Mealey et al., 2017).

On the contrary, some mitophagy-related proteins may also influence Nrf2 expression and activity. As a target gene of Nrf2, p62 competes with Nrf2 for binding to Keap1, thereby promoting Nrf2 evasion of Keap1-mediated ubiquitination and degradation of Nrf2 (Mizunoe et al., 2018). Transcriptome analysis of PINK1-deficient Drosophila showed that restoration of mitophagy by activating Nrf2 to upregulate both ribose pyrophosphate amidotransferase and mitochondrial methylenetetrahydrofolate dehydrogenase 2 could prevent PINK1 deficiency-induced neurotoxicity (Christensen and MacKenzie, 2008; Gumeni et al., 2021).

Nrf2 could also be activated by phagocytosis of Mst1/2 at the mitochondrial membrane. ROS release recruits Mst1/2 from the cytosol to the phagosome or mitochondrial membrane, where ROS subsequently activates Mst1/2 and phosphorylates the four Ser/Thr residues at the N-terminal end of Keap1, inhibiting Keap1 from forming a dimer and thus activating Nrf2 (Wang et al., 2019a).

Recent studies have demonstrated that treatment with NOX4 blockade or mitochondria-specific ROS inhibitors may activate the Nrf2 redox system, whereas increased levels of ROS and mtROS may enhance mitochondrial autophagy, leading to amelioration of cellular oxidative stress and toxicity (Fig. 4H) (Fan et al., 2023). Thus, the interactions between mitophagy and Nrf2 not only eliminate misfolded proteins and damaged mitochondria, but also ameliorate oxidative stress and inflammatory responses and reduce cytotoxicity.

Compounds acting through Nrf2–mitochondria interaction

Multiple mitochondrial activators also act on Nrf2. We previously defined some compounds such as α-lipoic acid, acetyl-l-carnitine, and coenzyme Q10 as mitochondrial nutrients, which effectively protect mitochondria from oxidative stress and improve mitochondrial biogenesis and respiratory complex activity. We and others reported that punicalagin may alter Nrf2 abundance by phosphorylating AMPK or PI3K/AKT sites (Cao et al., 2020a; Xu et al., 2016). Methylglyoxal activates Nrf2 and restores mitochondrial dysfunction by activating the PI3K/AKT site (Li et al., 2008).

Meanwhile, most of them could enhance Nrf2-mediated antioxidant enzyme system to treat aging-related neurodegenerative diseases and metabolic-related disorders (Liu and Ames, 2006). We summarized a series of compounds, which could ameliorate glucose or lipid metabolic disorders, oxidative stress or inflammatory damage through Nrf2–mitochondria interactions, as shown in Table 2.

We found that α-lipoic acid enhances intracellular GSH levels and increases phase II enzymes such as HO-1 and Γgcs expression to reduce ROS and RNS toxicity, and protects the integrity of mammalian DNA (Cvetko et al., 2021; Liu et al., 2009). Interestingly, supplementation with α-lipoic acid also promotes mitochondrial biogenesis in 3T3-L1 adipocytes (Zeng et al., 2021). Sulforaphane treatment induces PGC-1α and PGC-1β expression as well as mitochondrial mass in human fibroblasts, whereas PGC-1α is significantly reduced in fibroblast C2C12 from Nrf2 siRNA knockdown mice (Brose et al., 2012). We also found that punicalagin reduced blood triglycerides and ameliorated palmitate-induced lipotoxic inflammation via activation of Nrf2 and promotion of mitochondrial biogenesis and aerobic respiration (Yan et al., 2016).

Recently, our results showed that ω-3 polyunsaturated fatty acids (n-3) improved insulin resistance, mitochondrial oxygen consumption rate, and glycolysis in obese mice by downregulating the expression of TCA cycle enzymes IDH1, IDH2, SDHA, FH and MDH2, and mTORC1 signaling pathway (Liu et al., 2021).

In addition, MitoQ is reported to alleviate mitochondrial DNA depletion and defective mitotic phagocytosis in the kidney of db/db mice through upregulation of Nrf2 and PINK1 (Xiao et al., 2017). Melatonin could abrogate carbon ion-induced mitochondrial dysfunction in mice through coactivation of Nrf2 and PINK (Liu et al., 2018b). The above studies suggest that targeting the interactions between mitochondria and Nrf2 may be a novel strategy to treat metabolic-related disorders.

Obesity

As one of the most serious public health problems, there are ∼2 billion overweight and obese people worldwide, causing 4 million deaths annually (Bray et al., 2017). Nrf2 participates in balancing the expression of various adipogenic genes, including fatty acid-binding protein 4, CCAAT/enhancer-binding protein α/β (CEBPα/β), sterol regulatory element-binding protein-1c, PPARγ, fatty acid synthase, and acetyl CoA carboxylase (Wu et al., 2011).

Nrf2 is abundantly expressed or highly induced in human and mouse adipocytes, as well as in white adipose tissue (WAT). In addition, Nrf2 could effectively control WAT expansion and insulin sensitivity through the mitochondrial fatty acid synthesis pathway involving Htd2, maintaining glucose and lipid homeostasis in vivo (Xue et al., 2013; Zeng et al., 2021). Nrf2 deficiency strongly affects adipocyte differentiation. Studies in leptin-KO mice have shown that Nrf2 deficiency impairs CEBPβ and PPARγ protein levels, and disrupts adipocyte differentiation (Xue et al., 2013).

Brown adipose tissue (BAT) maintains body weight and metabolic status by regulating fatty acid synthesis and energy homeostasis (Schulz et al., 2007). Nrf2 activation modulates ATP generation by increasing mitochondrial biogenesis, and thus affects the involvement of BAT in energy metabolism by uncoupling proton motive force from OXPHOS through the action of uncoupling protein 1 (UCP1) (Holmstrom et al., 2013). In hepatocytes and adipocytes, Nrf2 may promote lipolysis and oxidation by upregulating lipid metabolism-related gene expression, such as PPARα and liver X receptor α, to reduce the blood lipid level (Kay et al., 2011).

In addition, Nrf2 may improve glucose metabolism in adipocytes by stimulating the expression of glucose metabolism-related genes such as PPARγ, hence preventing and treating T2D (Mohamed and Schaalan, 2018). Interestingly, systemic Nrf2 deficiency does not further worsen an already extremely obese phenotype, with systemic Nrf2 gene deletion attenuating hyperglycemia in mice and humans, and avoiding obesity (Chartoumpekis et al., 2011) and insulin resistance (Meakin et al., 2014) in HFD mice.

Notably, there are tissue differences in liver and adipocytes for Nrf2 deficiency. Adipocytes-specific Nrf2 KO HFD mice have elevated fasting blood glucose and fatty acid levels, with reduced WAT mass, leading to a worsened MetS, exacerbating insulin resistance, hyperglycemia, and hypertriglyceridemia (Xue et al., 2013). In contrast to adipocyte-specific Nrf2 KO mice, liver-specific Nrf2 KO mice tended to improve insulin sensitivity and decrease the inhibitory effect of Nrf2 on gluconeogenesis, but having little effect on hepatic triglycerides (Chartoumpekis et al., 2018). This may be due to the fact that adipocyte-specific Nrf2 KO resulted in enhanced expression of UCP1 and Sirt1, augmenting adipocyte capacity for glucose uptake (Bean et al., 2021). Whereas the lipid transport process in the liver is regulated by multiple pathways, such as mTORC2, along as a target of lipid metabolism regulation upstream of Nrf2 (Li et al., 2022b).

mTORC2 is also engaged in modulating fatty acid oxidation of PPARα, implying that Nrf2 is not a required target of hepatic lipid metabolism regulation (Zhang et al., 2022a). Nevertheless, mice with disrupted Nrf2 exhibit congenital intrahepatic shunting and may also develop conditions such as hypoglycemia and ketoacidosis or hepatocyte apoptosis (Skoko et al., 2014; Sun et al., 2021). Moreover, Nrf2 deficiency reduces HO-1, NQO1, SOD3, Gpx2, Gpx4, and TrxR expressions, and evokes mitochondrial dysfunction to promote the development of obesity (Xu et al., 2012).

Mitochondria play a key role in generating energy in the form of ATP through OXPHOS, producing cellular metabolic substrates, modulating lipid turnover, and controlling adipocyte production and adipokine secretion (De Pauw et al., 2009). AMPK has become an important target of both mitochondrial quality control and lipid and glucose metabolism. During the development of obesity, decreased AMPK activity impairs mitochondrial biogenesis and function, as well as inhibits CPT1-mediated β-oxidation of long-chain fatty acids (Ratner et al., 2015). Moreover, our recent study showed that AMPK may also act upstream of Nrf2 to ameliorate obesity-related oxidative stress.

Some compounds may target both Nrf2 and mitochondria to improve obesity. We found that HFD may lead to reduced levels of mitochondrial complex II and the production of alpha-butyric acid in the livers of obese rats, which impaired the cellular TCA cycle and further resulted in mitochondrial dysfunction. Supplementation of hydrogen-producing compound (coral calcium hydride) could simultaneously promote mitochondrial aerobic respiration and activate Nrf2-mediated phase II detoxifying enzymes expression to ameliorate HFD-induced obesity, glucose, and lipid metabolic disorders and hepatic steatosis (Hou et al., 2016), which is in line with our previous studies [i.e., the effects of zeaxanthin (Zou et al., 2014), oleuropein (Sun et al., 2017), lipoamide (Li et al., 2008), and α-tocopherol (Feng et al., 2010)]. Thus, in ameliorating the onset of obesity, modulating mitochondrial homeostasis may be an effective synergistic strategy in addition to Nrf2 activation.

More importantly, activation of Nrf2 may restore mitochondrial quality and function by increasing mitochondrial-related antioxidant and anti-inflammatory gene expression, helping to maintain glucolipid metabolic homeostasis and to protect from a range of risk factors associated with obesity.

Type 2 diabetes

The incidence and prevalence of diabetes continue to surge. The WHO predicts that diabetes-related mortality will double between 2005 and 2030, with T2D accounting for >90% of the cases (www.who.int/diabetes/en/). The major feature of T2D is hyperglycemia caused by insulin resistance. Our previous review has summarized the pivotal role of Nrf2 in insulin resistance (Liu et al., 2009). Nrf2 deficiency leads to elevated ROS and blood glucose levels, and impaired insulin sensitivity.

The mechanism by which Nrf2 ameliorates T2D may be through inhibition of the NF-κB promoter to resist inflammatory responses and through mediating downstream antioxidant systems to improve oxidative stress (Billeter et al., 2016; Guo et al., 2021). In addition, Nrf2 may have beneficial effects on diabetic complications. Compelling evidence from Nrf2 KO mice confirms the role of Nrf2 in the development of T2D-related kidney injury (Gupta et al., 2021; Li et al., 2020; Su et al., 2023).

Activation of Nrf2 may restore renal function in diabetic rats by normalizing protein expression of the downstream genes γGCS and HO-1, and significantly reducing extracellular matrix deposition and malondialdehyde concentration in glomeruli (Li et al., 2011a), as well as contribute to the improvement of antioxidant capacity in diabetic patients (Tan et al., 2011). Therefore, Nrf2 can significantly inhibit the onset of T2D and its complications, but the treatment of diabetes must be achieved by activating Nrf2 in a timely, highly specific, and coordinated manner to avoid side effects such as causing excessively low ROS levels (Matzinger et al., 2018).

In addition to Nrf2, mitochondrial dysfunction appears to play a central role in insulin resistance and T2D (Zhang et al., 2022b). Recently, we identified the importance of succinate dehydrogenase assembly factor 4 (SDHAF4), a novel assembly factor of mitochondrial II, in the regulation of hepatic insulin sensitivity through the arginine–NO pathway (Wang et al., 2022a). Furthermore, we reported Htd2, a key enzyme in mitochondrial fatty acid synthase pathway, played a vital role in the development of T2D. Deficiency of Htd2 resulted in triglyceride accumulation, oxidative stress, and impaired insulin sensitivity (Zeng et al., 2021).

Notably, mitochondria and Nrf2 may have synergistic effects in the development of T2D. As a major source of ROS production, increased mitochondrial respiratory complex activity may mitigate the side effects associated with low ROS level caused by Nrf2 activator (Schottlender et al., 2021). In diabetes, metformin acts on mTORC1 to trigger the downstream target ATF4. ATF4 activation facilitates FGF21 expression that enhances NADPH synthesis in mitochondria and activates the antioxidant effects of Nrf2 (Ait Ghezala et al., 2012; Kim et al., 2013; Torrence et al., 2021).

Furthermore, Keap1 downregulation and suppression of mtROS rescue Nrf2 expression to ameliorate diabetes (Kasai et al., 2020). In T2D rat models, activation of Nrf2 enhances cellular defense, reduces superoxide production, increases GSH and NADPH enzyme activity, and promotes mitochondrial biogenesis to prevent vascular oxidative stress (Van der Werf et al., 2018).

Some compounds may target both Nrf2 and mitochondria to prevent and treat T2D and its complications. Phosphocreatine could repair mitochondrial dysfunction via PI3K/Akt and reduce oxidative stress via Nrf2/HO-1 to treat T2D-related neurodegenerative disease through a dual target collaboration (Li et al., 2018). We recently reported that α-lipoic acid could promote mitochondrial biogenesis, improve mitochondrial aerobic respiration, and energy metabolism via the AKT/Htd2 cascade to improve insulin resistance (Zeng et al., 2021).

More importantly, in addition to upregulation of the expression of mitochondrial biogenesis-related proteins AMPK, PPARγ, PGC-1α, Tfam, and NRFs to promote mitochondrial biogenesis, we found that α-lipoic acid could simultaneously induce Nrf2-mediated phase II enzymes to ameliorate oxidative stress, inflammatory responses, and enhance insulin sensitivity to relieve the symptoms of T2D (Liu et al., 2009). These results are consistent with our previous studies on other compounds such as B vitamins, berberine, and EGCG (Liu et al., 2009).

Cardiovascular disease

CVD is the foremost medical problem in today's community and is another primary risk factor for MetS, mainly including myocardial ischemia/reperfusion injury, congestive heart failure, atherosclerosis, myocardial infarction, etc. Nrf2 activation has shown significant benefits against a diverse range of CVDs such as hypertension and cardiac hypertrophy by reducing ROS and upregulating the expression of antioxidant and detoxifying proteins (Bjorkman and Pereira, 2021), which has been reviewed recently by Chen (2022) and Chen (2021).

Nrf2 KO mice exhibited increased susceptibility to angiotensin II-induced cardiac hypertrophy and fibrosis as well as cardiac dysfunction (Li et al., 2011b). In addition, Nrf2 KO mice showed more vascular ROS production and more severe endothelial dysfunction induced by HFD (Cheng et al., 2011). Nrf2 also prevents CVDs by other mechanism other than the improvement of MetS-related risk factors. Activation of Nrf2 modulates lipid metabolism and vascular function by enhancing autophagy for the purpose of lowering blood pressure and improving vascular dynamics, thereby reducing atherogenesis (Lazaro et al., 2018).

In addition, activation of the Nrf2/ERK5 cascade reduces advanced glycosylation end products-induced vascular smooth muscle cell proliferation and ROS production to ameliorate vasculopathy from diabetes (Hwang et al., 2017). Mitochondria account for >30% of the total volume of cardiomyocytes and play a key role in determining the fate of cardiomyocytes (Ramachandra et al., 2020). It has been reported that impaired myocardial mitochondrial OXPHOS and mitochondrial biogenesis can lead to myocardial diastolic dysfunction (Prasun, 2020). Our recent data showed that mitochondrial complex II assembly factor SDHAF4 was essential in preventing dilated cardiomyopathy via modulating mitochondrial fission and mitophagy (Wang et al., 2022b).

Nrf2 and mitochondria may also be involved in the development of CVD through synergistic effects. The elevation of mitochondrial-derived ROS is an important cause of myocardial ischemia–reperfusion injury partially by opening the mitochondrial permeability transition pore (Mptp). Nrf2 could exert cardiac protection effect against ischemia–reperfusion injury by ameliorating mtROS-induced oxidative stress and inhibiting the opening of Mptp, as reviewed by Cadenas (2018). In aging-related CVD, Nrf2-mediated antioxidant pathway could preserve the mitochondrial integrity and mitochondrial OXPHOS function to improve myocardial dysfunction (Bose et al., 2020).

Some compounds may target both Nrf2 and mitochondria to prevent and treat CVD. We recently reported that punicalagin could improve hyperlipidemia-induced ED by promoting FoxO1-mediated mitochondrial biogenesis and reducing p38 MAPK/NF-κB–mediated inflammatory response (Liu et al., 2019). In an ischemia/reperfusion model, resveratrol could target mitochondria to improve antioxidant and inflammatory effects after myocardial ischemia by activating the Nrf2/ARE/PGC-1α signaling pathway (Dludla et al., 2017). We found that punicalagin could prevent HFD-induced cardiac metabolic disorders by activating AMPK to promote PGC-1α–mediated mitochondrial biogenesis and Nrf2-mediated antioxidant pathway (Cao et al., 2020a).

Consistently, our previous study showed that herba houttuyniae extract could also effectively activate AMPK/PGC-1α/Nrf2 cascade to protect against hyperlipidemia-induced myocardial injury (Cao et al., 2020b). Our previous data also revealed that curcumin could enhance mitochondrial biogenesis and respiratory complex activity, and activate Nrf2/MAPK/NF-κB pathway to inhibit downstream inflammatory response (Li et al., 2013).