Copper and hepatic lipid dysregulation: Mechanisms and implications

Abstract

Metabolic dysfunction-associated steatotic liver disease (MASLD) and Wilson's disease (WD) are common clinical conditions characterized by hepatic steatosis. Copper has been associated with the progression of hepatic steatosis, but its precise mechanism remains unclear. Emerging research on hepatic copper homeostasis-including its role in liver aging, cuproptosis induction, and lipid metabolism dysregulation-highlighted its significance in liver pathophysiology. Multiple mechanisms have been implicated in copper-induced hepatic lipid metabolism abnormalities, including oxidative stress, mitochondrial dysfunction, endoplasmic reticulum stress, impaired glucose metabolism, AMPK activation, hepatic nuclear receptor modulation, and cuproptosis. Both copper excess and deficiency could trigger hepatic steatosis via these pathways. This review systematically summarized intracellular copper metabolism regulation, elucidated potential mechanisms of copper-induced hepatic lipid dysregulation, and analyzed copper-lipid metabolism interactions in MASLD and WD. These findings provide insights into the mechanisms by which copper contributes to hepatic steatosis and offer a theoretical basis for targeting copper homeostasis as a therapeutic strategy in the treatment of liver diseases.

Key Words: Copper; Hepatic steatosis; Mechanism; Metabolic dysfunction-associated steatotic liver disease; Wilson's disease

Core Tip: Hepatic steatosis diseases are strongly associated with copper, and it has been shown that copper levels promote hepatic fat synthesis or lipolysis by triggering different mechanisms, which is extremely important for unravelling the molecular mechanisms of hepatic steatosis in diseases such as metabolic dysfunction-associated steatotic liver disease and Wilson's disease. This article reviews the potential mechanisms of copper-induced hepatic lipid dysregulation and explores new therapeutic targets for hepatic steatosis diseases.

INTRODUCTION

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common liver disease worldwide and a leading cause of liver-related disease and death, with pathogenic factors including obesity, type 2 diabetes mellitus (T2DM), hyperlipidemia, and metabolic syndrome[1]. As an essential trace element in the human body, copper is a key regulator of various physiological processes, and its dysregulated homeostasis can significantly impair normal liver function. Recent studies have shown that intracellular copper homeostasis in hepatocytes is not only involved in the regulation of liver aging but also influences the disease process by mediating cuproptosis, a novel form of regulated cuproptosis triggered by copper ion and copper ionophore[2,3]. Moreover, clinical evidence suggests that copper levels are associated with several metabolic disorders, including obesity, diabetes, and MASLD[4-6]. It has been suggested that hepatic copper is a potential etiological factor in hepatic steatosis and that cuproptosis may contribute to the development of MASLD[7,8]. Additionally, Wilson's disease (WD) is a typical disease of hepatic copper overload, in which the incidence of hepatic steatosis is as high as 50%[9]. Therefore, greater attention should be given to the critical role of copper in the liver, particularly in elucidating its mechanism of action on hepatic lipid metabolism. This will provide a new theoretical foundation and experimental direction for uncovering the molecular mechanisms of hepatic steatosis and developing intervention strategies based on copper homeostasis regulation.

However, current evidence regarding the effects of copper on hepatic lipid metabolism remains heterogeneous, suggesting involvement of multiple underlying mechanisms. Elevated hepatic copper regulates hepatic lipid metabolism bidirectionally by either inducing hepatic steatosis or promoting liver lipolysis[10,11]. In contrast, hepatic copper deficiency mainly promotes hepatic lipid synthesis[12]. Therefore, this paper provides a systematic overview of hepatic steatosis by reviewing the results of various studies, outlining the regulation of intracellular copper homeostasis and focusing on the potential mechanisms of copper-induced hepatic steatosis such as cuproptosis to gain insight into the mechanisms of hepatic steatosis in MASLD and WD.

COPPER METABOLISM AND ITS FUNCTION

Normal copper metabolism

Copper is an essential trace element required as a cofactor for the synthesis of copper-dependent enzymes, including copper/zinc superoxide dismutase (SOD1), ceruloplasmin (CP), cytochrome C oxidase (CcO), dopamine-β-hydroxylase, copper-containing amine oxidases, and tyrosinase. These enzymes play crucial physiological roles, including antioxidant defense, iron homeostasis, oxidative phosphorylation, and myelin sheath formation[13,14].

Copper absorption occurs mainly in the small intestine. The extracellular copper ions exist in the Cu(II) form. Although divalent metal transporter 1 can incorporate Cu(II), these cupric ions are not directly useable by cells. Most Cu(II) is reduced into Cu(I) by copper reductases such as DCYTB and STEAP. It is transported to the intestinal epithelial cells by the copper transport protein (Ctr1) and then excreted into the portal system by basolateral copper-transporting ATP7A, where it is transported in combination with albumin, macroglobulin, and histidine. Upon entering the liver, copper ions are transported by the copper-transporting ATP7B in hepatocytes to the trans-Golgi network (TGN) to synthesize holo-CP, which is secreted into blood circulation for distribution to tissues and organs. Excess copper in hepatocytes is transported via the ATP7B protein and excreted in bile[15-17].

Copper ions enter the cell via Ctr1 and have three main destinations: (1) Formation of copper-dependent enzymes mediated by copper chaperone proteins (CCS); (2) Secretion into the blood or bile; and (3) Intracellular storage. The SOD CCS supplies copper ions to SOD1 in the cytoplasm and mitochondrial membrane space (IMS); it also transports copper ions to the nucleus, activates the transcription factor HIF1, and increases the transcription of metallothionein (MT) genes in the presence of excess copper. The Cox17 Located in the IMS transfers cytoplasmic copper to the IMS, delivering it to CcO synthase (Sco1/2) and Cox11, respectively, both of which insert copper ions into CcO[18,19]. ATOX1 delivers copper ions to ATP7A and ATP7B proteins located in the TGN membrane, facilitating the synthesis of copper-dependent enzymes. For example, ATP7A-delivered copper is involved in lysyl oxidase and tyrosinase synthesis, while ATP7B delivers copper to CP[16,20,21]. CP and its homologue hephaestin are involved in iron oxidation in the reticuloendothelial system and intestinal epithelial cells, respectively[22]. ATOX1 also transports copper to the nucleus and functions as a copper-dependent transcription factor[23]. When intracellular copper levels increase, ATP7A/B proteins are localized in vesicles that fuse with the plasma membrane to excrete excess copper into the bile ducts or blood circulation. Upon restoration of physiological copper levels, ATP7A/B proteins are recycled back to the TGN[24]. Furthermore, AP-1 can recognize and package specific membrane proteins from the Golgi membrane into vesicles within the cell[25]. Additionally, it can bind to MT1/2 and glutathione (GSH) to prevent cytotoxicity when intracellular copper levels exceed its processing capacity[16]. The figure below shows the process of copper homeostasis regulation in the cells (Figure 1).

Figure 1 Regulation of copper homeostasis in cells. Copper metabolism is a complex and dynamic process that is regulated by multiple molecules at the cellular and organ levels. Copper uptake is mediated by Ctr1 and divalent metal transporter 1. Intracellularly, copper is transported to different subcellular organelles by various copper chaperone proteins, such as Cox17, CCS, and ATOX1, which facilitate the synthesis of copper-dependent proteins. When intracellular copper ions increase, they can bind to MT1, MT2, and GSH to limit the cytotoxicity of excess copper. ATP7A/B are trafficked to vesicles (AP-1 mediates normal vesicle formation), allowing the excretion of excess copper in hepatocytes into the gallbladder. Additionally, ceruloplasmin and its congener hephaestin participate in the normal oxidation of iron ions. This figure was created using FigDraw (www.figdraw.com) (Supplementary material).

Copper homeostasis and cuproptosis

In mammalian cells, two highly homologous copper-transporting ATPases, ATP7A and ATP7B, precisely regulate intracellular copper concentrations within the physiological range by hydrolyzing ATP. While both ATPases are expressed in the brain, developing kidney, placenta, mammary gland, eye, and lung, only ATP7B is expressed in hepatocytes[24]. Mutations in ATP7A cause Menkes disease by disrupting the activity of copper-dependent enzymes, including lysyl oxidase, tyrosine oxidase, and peptidyl α-amidating monooxygenases, resulting in neurodevelopmental abnormalities, connective tissue lesions, and defective vascular morphogenesis[26,27]. In WD, mutations in the ATP7B gene disrupt CP synthesis and biliary copper excretion, resulting in hepatic copper accumulation, neurotoxicity, and reduced serum copper levels[28]. Additionally, AP1S1 gene mutations impair AP-1 complex assembly, compromise ATP7A/B membrane localization, and cause MEDNIK syndrome-a multisystemic copper metabolism disorder with dermal-neural axis developmental abnormalities[25].

In 2019, Tsvetkov’s team identified the molecular mechanism of cuproptosis, revealing that excess Cu²⁺ triggered proteotoxic stress by binding to lipid-acylated proteins in the tricarboxylic acid (TCA) cycle, leading to protein aggregation and iron-sulfur cluster (ISC) loss[29]. Unlike apoptosis or ferroptosis, this pathway plays a role in copper metabolism disorders[3]. Evidence from WD mouse models, including reduced lipoylated proteins, depleted ISCs, and Hsp70 upregulation, suggested cuproptosis contributed to multiorgan damage[29]. In cardiovascular diseases, copper imbalance exacerbates tissue injury through oxidative stress (OxS), proteasome inhibition, and cuproptosis activation[30]. This discovery also advanced cancer therapy, with copper ionophores (e.g., elesclomol, disulfiram) selectively inducing cuproptosis in tumor cells[31].

Clinical studies of copper and lipid metabolism disorders

Copper is closely linked to the development of lipid metabolic diseases, and understanding its regulatory mechanisms is an important research focus. Elevated serum copper levels are associated with multiple cardiometabolic risk factors, including dyslipidemia, T2DM, obesity, and MASLD, which are potential disease predictors[32]. Cohort studies of United States adults demonstrated that high serum copper was associated with elevated serum concentrations of total cholesterol (TC) and high-density lipoprotein (HDL) cholesterol and was associated with higher risks of high TC and high low-density lipoprotein cholesterol (LDL-C)[33]. These findings are consistent with those of meta-analyses, which reported significantly higher serum copper levels in obese adults and children compared to healthy-weight controls, with higher copper levels associated with increased prevalence of obesity in the United States[34,35]. A Mendelian randomization analysis found that obesity mediated the association between serum copper and inflammation, and high serum copper could be a cause of obesity[36].

Dietary copper intake also exerted dose-dependent effects on lipid metabolism. A nationwide Chinese cohort study revealed a U-shaped relationship between dietary copper intake and obesity risk, indicating that both excessive and insufficient intake increased general and abdominal obesity risks[37]. For instance, higher copper intake elevated the prevalence of hypertriglyceridemia, particularly among United States adolescents with a body mass index ≥ 23 kg/m²[38]. Conversely, another study reported inverse associations between dietary copper and fasting glucose, TC, and LDL-C[39]. In rats, moderate copper supplementation reduced triglyceride (TG), TC, and LDL-C levels, although it did not significantly affect body weight, food intake, or HDL[40]. While copper deficiency was associated with adverse lipid profiles, supplementation was not universally recommended due to its potential pro-inflammatory and pro-oxidative effects[39].

However, inconsistencies in findings may be attributed to geographical, demographic (e.g., age, sex), and lifestyle (e.g., exercise, smoking, alcohol) variations that influenced copper-lipid metabolism interactions[41]. For instance, a European study found no correlation between copper levels and body fat percentage in children, whereas obese Indian children exhibited reduced serum copper and zinc levels[42,43]. Methodological differences in copper measurement and control group selection may have introduced additional bias[6]. These discrepancies suggest that copper played a dual role in lipid metabolism, with its biological effects dependent on homeostatic balance and individual metabolic regulation.

POTENTIAL MECHANISMS OF COPPER-INDUCED LIPID DYSREGULATION

The effects of copper on hepatic lipid metabolism include modulating lipoprotein synthesis, regulating de novo lipogenesis (DNL) and fatty acid β-oxidation (FAO), and disrupting glucose homeostasis. The pathogenic mechanisms will be elaborated below.

Copper and OxS and mitochondrial dysfunction

Excessive intracellular copper levels increase the generation of reactive oxygen species (ROS) and trigger OxS through multiple mechanisms. These included: (1) Copper-catalyzed ROS production via Fenton-like reactions; (2) GSH depletion under high copper exposure; and (3) Mitochondrial dysfunction due to copper toxicity and ROS disrupt membrane potential, impair electron transport chain activity, and exacerbate ROS. Notably, copper deficiency increases ROS and OxS by reducing the activity of antioxidant enzymes such as SOD1 and CcO[44,45].

Copper-induced ROS and OxS play a key role in hepatic lipogenesis through several mechanisms: (1) Generation of cytotoxic metabolites (e.g., malondialdehyde, lipid peroxides, 8-isoprostane, and 4-hydroxy-2-nonenal) via OxS-mediated lipid peroxidation, leading to cellular membrane damage[46]; (2) Activation of JNK and SREBP pathways by ROS, stimulating lipogenesis and lipid droplet (LD) formation. LDs may serve as a cellular defense mechanism against OxS[47,48]; (3) At the molecular level, Nrf2 acts as a central regulator. Copper overload-induced ROS activates Nrf2, promoting its nuclear translocation and binding to the PPARγ promoter, thereby upregulating PPARγ-mediated lipogenesis. Additionally, copper upregulates Nrf2 expression by enhancing MT transcription factor 1 binding to the Nrf2 promoter while inhibiting the SP1/Fyn pathway, further amplifying Nrf2-driven lipogenic gene transcription and TG deposition[49-51]; (4) ROS impairs mitochondrial integrity, disrupting oxidative phosphorylation and FAO, a key mechanism in copper-induced steatosis[52]. Concurrently, copper toxicity and suppressed mitophagy exacerbate mitochondrial dysfunction[53]; and (5) ROS-mediated copper overload induces hypermethylation of the PGC-1α promoter, thereby repressing its expression. This leads to impaired mitochondrial FAO and hepatic insulin resistance (IR)[54,55].

In summary, copper disrupts lipid metabolism by inducing OxS and mitochondrial dysfunction, altering hepatocyte structure, activating the JNK/SREBP pathway, increasing Nrf2 nuclear accumulation, and impairing FAO, ultimately promoting hepatic steatosis.

Copper activates endoplasmic reticulum stress

The endoplasmic reticulum (ER) is essential for hepatocytes to maintain lipid metabolism, with its regulatory roles including: (1) ER membrane-localized sterol regulatory element-binding proteins (SREBP-1c, SREBP-2), which regulate DNL-SREBP-1c primarily mediates fatty acid synthesis, while SREBP-2 controls cholesterol synthesis; (2) ER-resident acyltransferases that catalyze hepatocellular TG synthesis; and (3) ER-mediated VLDL formation. Consequently, ER dysfunction is closely associated with hepatic steatosis[56].

Impaired protein-folding capacity due to adverse conditions results in misfolded protein accumulation, triggering ER stress and activating the unfolded protein response (UPR). The UPR involves three transmembrane ER-resident stress sensors: Inositol-requiring enzyme 1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6). Under normal conditions, these sensors remain inactive through binding to 78-kDa glucose-regulated protein (GRP78). However, during protein misfolding stress, GRP78 dissociates from ATF6, IRE1α, and PERK, activating the UPR to restore ER homeostasis[57].

ER stress induced by hepatic copper overload contributed to steatosis through multiple mechanisms: (1) OxS-mediated UPR activation: Intracellular OxS and ROS accumulation activate the UPR and ER stress[56]; (2) Epigenetic dysregulation of ER chaperones: Hypermethylation of Grp78 induced by ROS-mediated copper overload suppresses its mRNA expression and exacerbates ER stress[55]; (3) CREB-dependent lipogenic programming: Copper-activated CREB binding sites in the promoters of Grp78, Perk, Ire1α, and Atf6α, upregulating lipogenic genes[58] such as SREBP-1c, ACC, SCD1, fatty acid synthase (FAS); and (4) Insig-1 depletion and SREBP-1c activation: ER stress/UPR-induced depletion of Insig-1 promoted copper-dependent activation of hepatic SREBP-1c[59].

Collectively, copper overload activates SREBP-1c and upregulates lipogenic gene expression by inducing ER stress and Insig-1 depletion. Additionally, ER stress reduces ApoB secretion, impairing VLDL assembly and contributing to hepatic IR, thereby exacerbating hepatic steatosis[56,60]. Moreover, studies have demonstrated that, in addition to copper-induced OxS triggering ER stress, copper ions themselves can directly induce ER stress.

Dysregulation of glucose metabolism caused by copper

Clinical studies indicated that abnormal copper levels significantly contribute to the development of diabetes and its complications[4]. A prospective cohort study demonstrated that higher dietary copper intake was associated with an increased risk of T2DM[61]. Elevated plasma copper concentrations also correlated positively with impaired glucose metabolism and T2DM, an association potentially modulated by SOD1 gene polymorphisms[62].

The mechanisms underlying copper’s impact on glucose metabolism may involve the following pathways: (1) ROS-mediated IR: Copper-induced IR occurs through ROS generation, as evidenced by reductions in ROS levels and improvements in IR following copper chelator therapy[63]; (2) Pancreatic β-cell dysfunction: Elevated copper levels impair β-cell function, consistent with negative correlations between copper and homeostasis model assessment of β-cell function[64]. This effect may involve copper’s interaction with human islet amyloid polypeptide (hIAPP). While copper inhibits hIAPP fibrillization, it stabilizes toxic oligomeric aggregates, exacerbating β-cell degeneration in T2DM[65]; and (3) Hepatic farnesoid X receptor modulation: Farnesoid X receptor (FXR) activation suppresses glycolysis, enhances hepatic glycogen synthesis, and inhibits gluconeogenesis via intestinal FGF19 release[66]. However, copper exerts tissue-specific effects on FXR: Intestinal copper activates the FXR-FGF19 axis, whereas hepatic copper accumulation downregulates FXR expression[67,68].

In summary, copper dysregulation influenced glucose metabolism through IR, β-cell dysfunction, hIAPP aggregation, and FXR modulation. These findings highlight the extensive impact of copper on metabolic regulation. However, current research has focused on copper’s role in diabetic complications. Due to the limited research on copper-induced glucose metabolism dysregulation, it remains unclear whether such metabolic disturbances subsequently disrupt hepatic lipid metabolism. Further investigation is needed to elucidate this potential link.

Regulation of AMPK activity by copper

AMPK is a key regulator of cellular energy homeostasis, modulating metabolic pathways in response to intracellular AMP/ATP ratios. In lipid metabolism, AMPK inhibits fatty acid and cholesterol synthesis by phosphorylating and inactivating ACC and HMG-CoA reductase while enhancing catabolic processes such as glycolysis and FAO. Given these roles, AMPK activation has emerged as a potential therapeutic strategy for MASLD[69,70].

Current evidence indicates that both copper overload and deficiency can activate the AMPK signaling pathway, primarily through ATP depletion or ROS generation. Notably, copper-induced AMPK activation appears to be mediated through upregulation of the p-AMPK/AMPK ratio rather than through changes in AMPK or p-AMPK transcriptional levels[71-75]. In addition to the two prevalent mechanisms discussed above, a study also suggested that increased copper levels activate AMPK by promoting the assembly of the SCO1-LKB1-AMPK complex. SCO1, a copper chaperone of CcO, also functions as a scaffold protein that tethers the upstream kinase LKB1 to AMPK, facilitating AMPK phosphorylation. LKB1 is a master upstream kinase that directly phosphorylates and activates AMPK[11,76]. Importantly, copper-mediated AMPK activation stimulates catabolic processes, including FAO, mitochondrial biogenesis, and oxidative phosphorylation, thereby reducing hepatic lipid accumulation[11]. Another study found that copper-induced AMPK activation reduces intestinal fat accumulation in yellow catfish (Pelteobagrus fulvidraco) by downregulating the expression of lipogenesis-related genes (acca, fas) and lipid absorption-related genes (fatp4, fabp2 and CD36) while upregulating the expression of lipid transport-related genes (mtp, apoai and lpl)[77]. Additionally, copper-induced activation of AMPK in skeletal muscle has been shown to enhance the expression of FAO-related genes (e.g., cpt)[78].

However, Liu et al[79] reported contrasting findings in their study of hepatic lipid metabolism in Monopterus albus during chronic copper exposure. They observed significant downregulation of AMPK subunit transcripts (ampkβ1 and ampkγ1) and fatty acid catabolism-related genes (hsl, atgl), while lipid synthesis genes remained unchanged, ultimately leading to hepatic lipid accumulation.

This discrepant outcome may suggest two key implications. First, the mechanism by which copper overload leads to hepatic lipid accumulation (or reduction) involves multiple pathways and may be highly influenced by exposure duration and concentration. Supporting this interpretation, a previous study found that hepatic lipids in the javelin goby Synechogobius hasta exhibited concentration-dependent accumulation at 30 days of copper exposure, but demonstrated concentration-dependent reduction at 60 days of copper exposure[80]. Thus, despite AMPK activation, hepatic lipid accumulation may still occur. Second, copper-induced AMPK activation may exhibit concentration dependence. Studies on copper cytotoxicity have shown that relatively low copper concentrations induce AMPK activation, whereas higher concentrations suppress it[81].

Therefore, establishing a quantitative relationship between copper concentration, AMPK activation, and lipid metabolism is essential for clarifying these regulatory mechanisms.

Effects of copper overload on nuclear receptors

The nuclear receptor family in the liver includes PPARs, liver X receptor (LXR), and FXR, which can form heterodimers with RXR to participate in hepatic lipid metabolism, storage, transport, and elimination[82]. In adult and pediatric WD patients as well as Atp7b^-/- mice, elevated copper impairs hepatic nuclear receptor function, leading to reduced binding of FXR, RXR, HNF4α, and LRH-1 to promoter response elements and decreased mRNA expression of their target genes[67]. The downregulation of cholesterol biosynthesis in Atp7b^-/- mice was found to result from the inhibition of signaling by nuclear receptors, as evidenced by reduced expression of LXR/RXR targets such as FAS and HMG-CoA reductase. This reduced LXR/RXR activity may stem from decreased levels of endogenous activating ligands or lower expression of nuclear receptors[83,84].

The PPAR family comprises three isoforms: PPARα, PPARβ/δ, and PPARγ. PPARα is highly expressed in metabolically active tissues such as the liver, heart, and skeletal muscle, where it plays a key role in regulating genes involved in FAO. In contrast, PPARγ is predominantly found in adipose tissue, where it governs adipocyte differentiation, and it is also expressed at lower levels in the liver[85]. PPARγ activation is related to fat synthesis and hepatic steatosis[86]. Within specific concentration and time ranges, copper overload promotes hepatic lipid accumulation and modulates PPAR expression levels. For instance, in a study on copper exposure and hepatic lipid metabolism in Synechogobius hasta, 30-day copper treatment increased hepatic lipid content in a dose-dependent manner, accompanied by decreased PPARα mRNA expression and elevated PPARγ mRNA levels. Similar patterns were observed in Takifugu fasciatus and Monopterus albus exposed to copper[79,80,87]. However, excessive or prolonged copper exposure may reduce hepatic lipid content. For instance, in Synechogobius hasta, prolonged copper exposure (60 days) led to a dose-dependent decrease in liver lipids despite upregulation of both PPARα and PPARγ mRNA levels. Furthermore, dietary copper supplementation in rabbits reduced hepatic lipid deposition while increasing PPARα expression[78,80,88].

In contrast, in copper exposure experiments conducted on yellow catfish Pelteobagrus fulvidraco, variations in copper concentration did not significantly alter hepatic PPARα and PPARγ mRNA expression levels despite inducing noticeable changes in hepatic lipid metabolism[59,89]. The difference in hepatic lipid accumulation may be attributed to levels of PPAR expression in the liver between animals. For instance, PPARγ expression was most abundantly expressed in the liver of javelin goby Synechogobius hasta but significantly lower in Pelteobagrus fulvidraco[80].

Although PPAR transcript levels are strongly associated with copper exposure, the regulatory mechanisms of PPAR under copper overload remain unclear. When studying the effects of copper overload on hepatic steatosis, it is essential to consider the animal model, copper concentration, and exposure duration, as these factors also influence the expression levels of hepatic PPARα and PPARγ. A study has found that excessive Cu²⁺ may disrupt PPAR/RXR heterodimer formation, thereby inhibiting the PPARα pathway. Additionally, the zinc finger domains of PPAR could be a key target for copper-mediated interference[79,90].

Role of copper in adipose tissue

Adipose tissue serves as the primary site of fat storage. Impaired or excessive adipocyte lipogenesis disrupts lipid metabolic homeostasis, potentially contributing to hepatic steatosis[91]. Elevated serum levels of the copper-dependent proteins semicarbazide-sensitive amine oxidase (SSAO) and CP in obese individuals were linked to increased copper levels, driven by upregulated Ctr1 and ATP7A expression in adipose tissue[92]. One study identified CP as a new lipocalin and was secreted by adipose tissue during obesity[93], suggesting a key role of adipose tissue copper in lipid metabolism.

Copper plays a critical role in regulating adipose tissue differentiation, lipogenesis, and lipolysis. During adipocyte differentiation, the Wnt/β-catenin pathway-essential for mesenchymal stem cell adipogenesis-was inhibited upon activation. Elevated copper levels in preadipocytes stabilized β-catenin, suppressing adipogenesis[94,95]. Adipocyte-specific ATP7A knockout in mice increased adipose copper content, resulting in age-dependent white adipose atrophy, IR, and hepatic steatosis[96].

Copper also modulates lipid metabolism via SSAO, a copper-dependent enzyme highly expressed in adipose tissue. Normal SSAO activity supports adipocyte differentiation and energy metabolism, while copper deficiency impairs SSAO function, promoting adipocyte hypertrophy and fat accumulation[97]. Regarding lipolysis, a classic study demonstrated that copper enhances fat lipolysis by binding to a critical conserved cysteine residue in phosphodiesterase 3B (PDE3B), which is responsible for cAMP degradation. This interaction inhibits PDE3B activity, leading to increased cAMP levels and subsequent activation of downstream target genes[98].

Overall, copper regulates multiple aspects of adipose tissue metabolism. Generally, elevated copper levels in adipose tissue reduce lipid accumulation, whereas copper deficiency exacerbates it. Given that obesity is a major contributor to hepatic steatosis, modulating copper homeostasis may represent a potential therapeutic strategy for improving lipid metabolism in obesity.

Other potential mechanisms

microRNAs: microRNAs (miRNAs) are a highly conserved class of endogenous single-stranded non-coding RNAs that inhibit gene expression. There was a review emphasized that miRNAs are involved in various processes of hepatic steatosis, including enhancing hepatic lipid uptake and promoting DNL, impairing lipid oxidation, reducing hepatic lipid export, as well as influencing hepatic glucose metabolism, autophagy, and ER stress-related cellular processes[99]. In Pelteobagrus fulvidraco, copper exposure downregulated miR-205, which appeared critical in copper-induced lipid metabolic dysfunction. Copper overload reduced miR-205 expression, elevated TG levels, and FAS activity. These effects were attenuated by a LXR antagonist. LXRα, as a potential target of miR-205, may be a key target of miR-205 and mediate copper-induced lipid metabolic disruption[100].

This research provides another perspective into copper-induced hepatic lipid metabolic disorders, specifically through epigenetic mechanisms. The miRNAs are involved in multiple processes of hepatic steatosis, which establishes a theoretical foundation for future studies on miRNAs-mediated mechanisms underlying copper-induced liver steatosis.

Hepatic iron overload: Hepatic iron overload disrupts systemic lipid homeostasis via multiple pathways, including impaired insulin signaling, OxS, mitochondrial dysfunction, and activation of the HIF1α-PPARγ pathway[101-103]. Clinical studies have observed hepatic iron overload in patients with hepatic steatosis, which correlates with diminished ferroxidase activity of copper-containing proteins due to reduced hepatic and serum copper levels[104]. Additionally, iron metabolism abnormalities have been documented in some patients with WD[105]. Although both hepatic copper and iron dysregulation influence lipid metabolism, their mechanisms operate independently[106]. Hepatic iron overload may provide valuable insights into the effects of copper on hepatic lipid metabolism.

Wnt/β-catenin signaling pathway: Classical Wnt signaling in hepatocytes plays a crucial role in the development of diet-induced hepatic steatosis and obesity[107]. A study demonstrated that hepatic β-catenin deficiency impaired mitochondrial FAO, resulting in hepatic lipid accumulation. This suggests that the Wnt/β-catenin signaling pathway may modulate lipid metabolism by regulating sirtuin 1 (Sirt1) or PPAR-α expression. Sirt1, a NAD⁺-dependent deacetylase, is a member of the sirtuin family[108,109]. In grass carp hepatocytes, copper overload induces hepatic lipid deposition by suppressing the Wnt/β-catenin pathway, reducing nuclear β-catenin accumulation. Copper also inhibits SIRT1 mRNA expression, increasing β-catenin acetylation and enhancing lipogenic enzyme gene promoter activity[110]. However, a recent study found that copper exposure downregulated the expression of HIF-1α and β-catenin proteins in bovine mammary epithelial cells. This suppression subsequently leads to decreased expression of their downstream targets SREBP1 and PPARγ proteins and inhibited lipid synthesis[111]. This discrepancy may be attributed to differences in cell types, but more importantly, it suggested that copper likely affects the Wnt/β-catenin signaling pathway through distinct mechanisms.

Although the Wnt/β-catenin pathway regulates various hepatic pathophysiological processes, its involvement in copper-induced hepatic steatosis remains unclear. Thus, investigating whether copper influences β-catenin expression could serve as a feasible starting point for further research.

Activation of autophagy: Autophagy is a conserved process involving the sequestration and degradation of cytoplasmic components via autophagosomes to maintain intracellular homeostasis[112]. Dysregulated autophagy disrupts cellular homeostasis and contributes to hepatic steatosis, and autophagy activation appears to be a critical therapeutic target for alleviating hepatic steatosis[113,114]. Copper overload induces OxS, which modulates autophagy through multiple pathways, including inhibition of the PI3K/AKT/mTOR pathway, activation of the AMPK-mTOR axis, or direct suppression of mTOR signaling[72,115,116]. Additionally, the p38 MAPK pathway participates in copper-mediated autophagy regulation[117]. In lipid metabolism, autophagy plays a protective role against copper-induced lipid deposition[51]. However, one study reported that activation of autophagy enhanced lipid production in microalgae under Cu²⁺ stress[118]. Beyond species-specific differences, the finding provided another perspective into the interplay between autophagy and copper-regulated lipid metabolism.

Collectively, the four proposed mechanisms-miRNAs alterations, excess hepatic iron, aberrant Wnt/β-catenin signaling pathway, and activation of autophagy-all play important roles in lipid metabolism. However, current evidence supporting their involvement in copper-induced hepatic steatosis remains limited. Thus, systematically elucidating these mechanisms could provide novel insights for future research.

LIVER COPPER AND LIPID DYSREGULATION DISEASES

MASLD and copper

MASLD is a chronic liver condition defined as abnormal lipid accumulation in hepatocytes and is diagnosed as steatotic liver disease when hepatic fat content reaches ≥ 5%, as measured by proton density fat fraction via magnetic resonance imaging. The global prevalence of hepatic steatosis has increased significantly owing to rising rates of obesity, diabetes, and metabolic syndrome. As an early indicator of hepatic dysfunction, steatosis could progress to steatohepatitis, fibrosis, cirrhosis, and even hepatocellular carcinoma, posing a serious health risk[119,120]. Emerging evidence indicates that copper plays a significant role in MASLD pathogenesis, although the precise mechanisms remain complex and debated.

Many studies have consistently reported lower hepatic copper levels in MASLD patients, which are negatively correlated with the severity of hepatic steatosis[121]. Hepatic copper deficiency has been suggested as a potential factor in hepatic steatosis, supported by the findings of several studies. In obese patients, hepatic copper levels were significantly higher in those with no or mild steatosis and significantly lower in those with severe steatosis than in controls[92]. Additionally, research showed significantly lower concentrations of copper in the dry, defatted liver tissues of ob/ob obese mice. This study provides compelling evidence of true hepatic copper deficiency, independent of volume effect, as hepatic copper levels were already significantly reduced by the age at which ob/ob mice exhibited signs of metabolic liver disease[122]. Research involving dietary copper restriction in animals has shown that a low-copper diet results in decreased copper levels in the liver and causes hepatic steatosis[12]. Consistent with these findings, hepatic copper overload was found to reduce fat accumulation in MASLD by activating AMPK.

Hepatic lipid metabolism may play a role in regulating hepatic copper levels. Future rigorous prospective studies are necessary to establish the causal relationship between hepatic copper and hepatic steatosis. Studies have found that mice fed a high-fat diet (HFD) exhibit reduced hepatic copper levels, which may be associated with increased synthesis of the major copper exporting proteins (ATP7A and ATP7B) and CP[11,123], in addition to inhibition of intestinal copper absorption by the high fructose component of the HFD[124]. In vitro studies revealed that fatty acid uptake in hepatocytes induced early mitochondrial dysfunction, elevated cytoplasmic copper, and activated export mechanisms, reducing intracellular copper[125]. Conversely, some studies reported elevated serum and hepatic copper levels in HFD-fed mice, correlating with increased CTR1 and ATP7B mRNA expression[126].

Although MASLD is associated with hepatic copper deficiency, the exact molecular mechanisms remain unclear. Early animal studies suggested that copper deficiency promotes the rate of hepatic fatty acid synthesis and assembly into triacylglycerol and phospholipids[127]. Copper deficiency also enhanced cholesterol synthesis via elevated HMG-CoA reductase activity, raising plasma cholesterol and TG levels[128]. Conversely, hepatocellular cholesterol levels decreased due to reduced cholesterol 7-hydroxylase expression and increased Apo (B and A1)-mediated lipoprotein secretion[129-131]. Additionally, copper-deficient rats exhibited higher nuclear SREBP-1 Levels, attributed to reduced hepatic cholesterol and elevated GSH, which stimulated FAS expression and inhibited sphingomyelinase, enhancing SREBP-1 proteolytic release[130].

Several key issues require attention in contemporary research on MASLD and hepatic copper levels. First, it is essential to clarify whether decreased hepatic copper levels are a causative factor in MASLD or a secondary consequence of hepatic steatosis, as this distinction is crucial for understanding the mechanisms of the disease. Second, although patients with MASLD typically have lower copper levels in the liver, serum copper levels are not necessarily reduced[132-134]. This discrepancy underscores the need for further investigation into the role of hepatic copper in MASLD and for the development of more reliable serum biomarkers to assess copper status. Third, greater emphasis should be placed on the importance of maintaining hepatic copper homeostasis in preserving liver health and treating liver diseases. A previous study suggested that the reduction of hepatic copper in MASLD was associated with increased CP synthesis. Experimental CP ablation demonstrated restored copper levels and improved hepatic steatosis in MASLD mice, indicating that CP may be an important therapeutic target for MASLD[11]. Moreover, a recent study developed bio-friendly copper ionophores (HQFs) and showed that HQF-mediated copper delivery can safely and effectively mitigate the progression of fatty liver in mice[135]. Additionally, hepatic copper dysregulation plays a significant role in hepatocyte aging, and it is proposed that reducing overall copper intake or increasing antioxidant consumption may be an important strategy to promote liver longevity and counteract the effects of aging. Dietary copper intake levels are strongly associated with lipid metabolism. Thus, maintaining the delicate hepatic copper balance should be a key focus in future studies.

WD and lipid dysregulation

The chief pathological feature of WD was significant hepatic steatosis, with hepatic copper levels positively correlating with the degree of steatosis[136]. Hepatocyte-specific inactivation of the ATP7B gene confirmed that steatosis resulted directly from copper overload[10]. Factors such as patient age, ATP7B mutation type, and variants in the PNPLA3 gene, which encodes a triacylglycerol lipase, also influence the severity of steatosis. The PNPLA3 G allele and the age of the child were independent correlates of moderate and severe steatosis. Patients with a pure mutation in the ATP7B gene exhibited significantly lower hepatic fat content compared to those with other mutations. Conversely, patients with a mutation in exon 14 demonstrate a notably higher proportion of hepatic LDs than patients with mutations in other exons[9].

Studies on hepatic copper show that copper overload can lead to OxS, mitochondrial dysfunction, ER stress, abnormalities in glucose metabolism, dysregulation of AMPK, regulation of the Wnt/β-catenin pathway, activation of autophagy, alterations in miRNA, and modulation of nuclear receptors, all of which contribute to hepatic steatosis. Similar pathological changes can also be observed in WD patients and animal models[137-143]. However, WD reveals more complex mechanisms. Nuclear receptors (FXR, RXR, LXR) and their target genes were suppressed in WD[67,144], while PPARs exhibited dynamic changes: PPARα mRNA initially increased but declined with worsening liver injury, correlating with antioxidant enzyme activity. In contrast, whereas PPARγ mRNA expression gradually increased with the aggravation of liver injury and rose in parallel with the increase in hepatic steatosis score, it was speculated that PPARγ might promote the progression of steatosis[145]. Furthermore, the alterations in DNA methylation observed in WD patients appear to be associated with lipid metabolism. Gene Ontology analysis of WD-specific hepatic differentially methylated regions (DMRs) reveals that genes associated with hypermethylated DMRs are enriched in acute inflammatory response, lipid catabolism, and folate metabolism. In contrast, genes linked to hypomethylated DMRs are enriched in humoral immunity, fatty acid transport, and glycolytic regulation[146]. Although miRNA expression is regulated in WD patients, it may influence copper homeostasis rather than lipid metabolism[147]. Most studies focused on copper-induced liver injury, whereas investigations into the pathogenesis of hepatic steatosis in WD remain notably scarce. However, a zebrafish WD model implicated HIF-1 signaling disruption in steatosis pathogenesis[148]. Moreover, a study found significant differences in hepatic lipid metabolism between WD mice and hepatocyte-specific ATP7B knockout mice, implying that copper homeostasis of non-parenchymal liver cells and MT expression have potential effects on hepatic steatosis in WD[10]. Additionally, it has been suggested that Atp7b^-/- mice have decreased copper levels and decreased lipolysis in adipose tissue, which may also be potential factors affecting liver lipid metabolism[98].

Beyond hepatic steatosis, WD also alters serum lipid profiles. While most WD patients had normal serum cholesterol and TG, those with hepatic manifestations exhibited reduced cholesterol levels[149]. Similarly, WD mice showed decreased serum TG, likely due to the upregulation of lipoprotein lipase[150]. In WD mice, hepatic copper accumulation suppressed SREBP-2 and HMG-CoA reductase expression, downregulating cholesterol biosynthesis and potentially explaining the hypocholesterolemia[151]. Notably, this cholesterol reduction was likely a metabolic adaptation rather than a direct driver of steatosis[152].

Despite significant progress, several important questions regarding WD remain unresolved. First, in clinical practice, attention should be paid to the differential diagnosis between WD and MASLD to avoid delaying the treatment of WD. One study found that some WD patients were misdiagnosed with MASLD; subtle histologic features such as distribution and type of steatosis may help differentiate WD from MASLD. Consequently, it is necessary to test serum CP and 24-hour urinary copper to exclude WD in cases of unexplained hepatic steatosis[153,154]. Second, currently, the main therapeutic agents are copper chelators, such as D-penicillamine, zinc salts, and trientine[155]. However, a previous study demonstrated that LXR activation in Atp7b^-/- mice improved liver function and lipid metabolism despite hepatic copper still accumulating[84]. The finding provides a potential alternative therapeutic strategy for WD. Third, in addition to dietary copper restriction for WD, high-calorie dietary intake should also be considered. A study demonstrated that a high-calorie diet significantly exacerbates hepatic mitochondrial dysfunction and hepatocellular injury in WD rats, accelerating disease onset and progression[156]. Conversely, other studies have shown that a HFD appeared to ameliorate hepatic steatosis and inflammation in WD mice[140,152]. Further studies are needed to evaluate the necessity of dietary calorie restriction in WD patients.

CUPROPTOSIS IN WD AND MASLD

In WD patients, copper overload causes ISC damage, and significantly lower levels of lipoylase and ISC proteins were observed in WD mice compared to wild-type mice, suggesting a potential role of cuproptosis in WD pathogenesis[157]. ISC proteins are critical for enzymes in the TCA cycle and mitochondrial respiratory chain complexes I-III[158]; their impairment could disrupt metabolic homeostasis. Although no direct evidence links cuproptosis to hepatic steatosis, copper overload may influence lipid metabolism through OxS, mitochondrial dysfunction, IR, and dysregulated lipid-signaling pathways, implying a possible association[8].

Bioinformatics analyses have revealed that cuproptosis-related genes (CRGs) were involved in multiple pathological processes associated with MASLD, including glucose metabolism, lipid metabolism, OxS, and TCA cycle regulation. Among these, 13 CRGs were upregulated, and six were downregulated[159]. Wu et al[160] reported increased expression of DLD (affecting redox homeostasis) and PDHB (promoting pyruvate entry into the TCA cycle) in both MASLD patients and mouse models, suggesting their potential role in hepatic steatosis progression via cuproptosis. Similarly, Ouyang et al[161] identified three key CRGs-NFE2 L2 (attenuating hepatic steatosis and OxS), POLD1 (linked to lipid deposition), and DLD-and developed a diagnostic model for MASLD based on these genes. Additionally, Liu et al[162] identified three CRGs (ENO3, SLC16A1, and LEPR) involved in glycolysis, lactate transport, TG metabolism, and energy homeostasis, which could serve as predictive markers for MASLD risk.

These studies suggest that cuproptosis may play a significant role in hepatic steatosis. Interestingly, despite the reduced hepatic copper levels observed in MASLD patients, the expression of certain CRGs was found to be elevated. This finding highlights the complexity of copper metabolism in hepatic steatosis and indicates the need to reconsider its mechanistic role.

CONCLUSION

Copper is a key trace element with a tightly regulated metabolism. Clinical studies have demonstrated that copper metabolism disorders are closely linked to lipid metabolism diseases, including obesity, dyslipidemia, diabetes, and MASLD. Previous studies have proposed several mechanisms through which copper modulates hepatic lipid metabolism, such as OxS, mitochondrial dysfunction, ER stress, impaired glucose metabolism, AMPK activation, PPARs regulation, miRNA expression alterations, iron overload, Wnt/β-catenin signaling inhibition, and autophagy activation. Additionally, copper influenced hepatic lipid metabolism indirectly via adipose tissue. The discussion of MASLD and WD further elucidated the intricate link between copper, cuproptosis, and lipid metabolism.

However, key questions remained unresolved: (1) Most mechanistic insights were derived from aquatic animal studies, necessitating validation in clinical settings; (2) Copper exhibits dual regulatory effects on hepatic lipid metabolism, promoting both lipolysis and lipogenesis. However, uncertainties persist regarding how copper regulates hepatic steatosis; (3) The contributions of cuproptosis and other pathological mechanisms to hepatic steatosis in WD and MASLD require further direct evidence; and (4) Current research still lacks comprehensive strategies for effectively regulating copper homeostasis for disease treatment. In addition to the significant potential of cuproptosis in cancer therapy, restoring hepatic copper homeostasis may represent a critical therapeutic target for liver diseases, particularly in preventing hepatocyte senescence and steatosis. Future studies should prioritize elucidating copper’s specific role in hepatic steatosis in WD and MASLD, investigating trace element effects, and identifying therapeutic targets or diagnostic markers.