Introduction

Adipose tissue is not only an energy storage organ, but also an important endocrine organ. By secreting various bioactive substances called fat factors, it helps to regulate the whole body’s metabolic homeosis. Its abnormal function is closely related to metabolic disorders such as obesity and type 2 diabetes mellitus (T2DM).1 Neuregulin 4 (Nrg4) is a new fat factor discovered in recent years. It is an extracellular ligand of the epidermal growth factor (EGF) family. It is expressed in multiple organs, among which the highest expression level is in brown adipose tissue (BAT).2 A large number of studies have shown that Nrg4 levels in adipose tissue and serum decrease significantly with age, indicating a potential association with age-related metabolic diseases. This makes it a potential target for the treatment of obesity, type 2 diabetes and its vascular complications and other diseases.1 In animal studies, mice overexpressing Nrg4 showed significantly increased insulin sensitivity and significantly reduced liver fat degeneration when fed a high-fat diet. In addition, the level of Nrg4 in human fat tissue is negatively correlated with the content of body fat and liver fat.3 Although previous studies have shown that decreased Nrg4 expression in adipose tissue may be associated with obesity and impaired glucose tolerance,4 the exact mechanism of Nrg4’s role in human beings is still not fully understood. Nrg4 activates the downstream signalling pathway by binding to the ErbB receptor family (especially ErbB4).3 It is involved in regulating liver metabolism, glycolipid metabolism, inflammatory response, autophagy and mitochondrial function. Nrg4 can also be used in the hypothalamus and other central nervous systems through endocrine machines to regulate energy balance and metabolic homeostasis.5 The relationship between Nrg4 and insulin resistance is still controversial, and the results between clinical research and animal experiments contradict each other. For example, some studies reported a decrease in plasma Nrg4 levels in newly diagnosed patients with type 2 diabetes,6 while other studies showed a negative correlation between human serum Nrg4 levels and insulin sensitivity.7

This review provides a comprehensive overview of the physiological characteristics, receptors, and signaling pathways of Nrg4, with a focus on its mechanisms for improving insulin resistance, therapeutic potential, safety, and future research directions. To identify relevant literature, we searched PubMed and Web of Science using keywords including “neuregulin 4”, “Nrg4”, “insulin resistance”, “ErbB4”, “metabolic syndrome”, “autophagy”, and “inflammation” and its various forms. By integrating current evidence, this review aims to serve as a comprehensive reference for further elucidating the role of Nrg4 in metabolic diseases and its potential clinical applications.

Physiological Properties of Nrg4 Molecular Structure and Expression/Secretion of Nrg4

Nrg4 is a member of the small protein family containing epidermal growth factor (EGF) domains. It is synthesised as a transmembrane precursor, and its N-terminus contains extracellular EGFL domains. After protein hydrolysis is released outside the cell, it plays its biological role by activating the ErbB receptor.8,9 The primary structure of Nrg4 is unique, and its EGF-like domain is a key structural element that binds to ErbB receptors and activates downstream signalling pathways.10 Nrg4 shows specific expression patterns in the body. In mice, Nrg4 is expressed in the lung, heart, and adipose tissues, with the highest expression in brown adipose tissue (BAT).2 In human tissues, Nrg4 was first detected in the pancreas and muscles, and then, through DNA sequencing, was found to be expressed in 27 tissues, also with the highest expression in brown fat tissue.11 It should be noted, however, that Nrg4 expression levels are dynamically regulated; for example, under chronic cold acclimation, Nrg4 mRNA is markedly upregulated in inguinal white adipose tissue of mice, whereas its expression in BAT remains relatively low. Thus, tissue-specific expression patterns depend on species, metabolic state, and environmental conditions. Nrg4 mRNA expression is detectable in the adult pancreas, with weaker expression in muscle and no detectable expression in other tissues.10

The expression of Nrg4 is regulated by multiple factors, with temperature being a key modulator. Studies have revealed that under chronic cold acclimation (CA; 10°C) conditions, Nrg4 mRNA expression is significantly upregulated in mouse inguinal white adipose tissue (iWAT), while expression levels remain low in brown adipose tissue and epididymal white adipose tissue (eWAT). In comparison, Nrg4 expression in mouse liver showed no significant difference between thermoneutral (TN) temperature (30°C) and room temperature (RT; 25°C) conditions.12 This phenomenon suggests that Nrg4 may be involved in the body’s adaptation to cold environments and in the regulation of heat production. Chronic inflammatory infiltration of adipose tissue significantly downregulates Nrg4 expression in adipocytes through pro-inflammatory cytokines such as tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β).3 In inflammatory bowel disease (IBD) mouse models and human samples, Nrg4 levels are also markedly reduced.13 While Nrg4 overexpression can inhibit inflammatory responses by reducing macrophage infiltration and pro-inflammatory cytokine levels, forming a negative feedback regulation, metabolic status also affects Nrg4 expression. In adipose tissue of obese rodent models and obese humans, Nrg4 expression levels are significantly reduced.3,5 During glucose metabolism, cAMP activators significantly induce Nrg4 expression in primary mouse hepatocytes.11 Exercise may also influence Nrg4 expression. In one study, plasma Nrg4 levels significantly increased in 44 obese men after 12 weeks of resistance training.14

Nrg4 Receptors and Signaling Pathways

The receptor for Nrg4 belongs to the ErbB/HER receptor tyrosine kinase family, which comprises four receptors (ErbB1-ErbB4) that bind growth factor ligands containing EGF-like motifs.15,16 Nrg4 exhibits strict specificity for ErbB4. Synthetic peptides of Nrg4 containing the full-length EGF-like domain can induce growth in cells expressing ErbB4 ectopically.17 ErbB4 is a glycoprotein composed of a glycosylated extracellular domain (ECD), a single transmembrane domain, and an intracellular domain (ICD) containing the functional tyrosine kinase and C-terminal tail.11 The formation of dimeric complexes is crucial for ErbB4 function.11,18 ErbB4 can form homodimers or heterodimers with ErbB1 or ErbB2.19,20 ErbB2 has no known ligands but serves as the preferred heterodimerization partner for other ErbB receptors.21 ErbB3 lacks intrinsic kinase activity and only becomes functional upon heterodimerization with ErbB4.21 Nrg4 is initially synthesized as a type I transmembrane precursor protein with an extracellular EGF‑like domain. Proteolytic cleavage of the extracellular domain, likely by members of the ADAM family of metalloproteases, releases a soluble biologically active fragment containing the EGF-like domain, which then binds to ErbB4 receptors on neighboring or distant cells,22,23 inducing ErbB4 receptor dimerization and activating tyrosine kinase activity. This subsequently mediates its biological functions through pathways such as PI3K/Akt and STAT5.3,24

After binding to the ErbB receptor, Nrg4 will trigger receptor tyrosine phosphorylation, which in turn activates multiple downstream signaling pathways, including PI3K/AKT signaling pathway, MAPK (Erk-1/2) signaling pathway, STAT5 signaling pathway, AMPK/mTOR signaling pathway, etc, and participates in the regulation of cell metabolism, proliferation, apoptosis, inflammation, and other physiological processes. The PI3K/AKT signaling pathway is one of the key pathways mediated by Nrg4. Upon binding to the CYT-1 subtype of the ErbB4 receptor, Nrg4 activates PI3K activity, thereby promoting AKT phosphorylation while simultaneously reducing c-Jun N-terminal kinase (JNK) phosphorylation.11 The ability of ErbB4 to activate the PI3K/AKT pathway is largely restricted to the CYT-1 isoform, which contains a docking site for the p85 subunit of PI3K. The CYT-2 isoform, which lacks this site, does not effectively couple to PI3K and preferentially signals through other pathways such as MAPK. Therefore, the metabolic effects of Nrg4 via PI3K/AKT are isoform-dependent and may vary across cell types expressing different ErbB4 splice variants. Activated AKT regulates multiple metabolic processes by phosphorylating downstream substrates. It promotes the translocation of GLUT4 glucose transporters to the cell membrane, inhibits transcription factors such as GSK3β and FoxO1, increases glucose uptake, enhances glycogen synthesis, and reduces hepatic glucose production.25,26 Nrg4 inhibits JNK phosphorylation, thereby downregulating the gene expression of downstream inflammatory mediators such as IL-1β and TNF-α. This reduces inflammatory responses and consequently alleviates insulin resistance.1,11 Nrg4 activates AMPK both in vivo and in vitro, inhibits mTOR phosphorylation, promotes LC3B-II conversion, reduces P62 accumulation, enhances autophagic flux, decreases lipid accumulation in hepatocytes, lowers serum and hepatic triglyceride (TG) and total cholesterol (TC) levels, and improves insulin resistance.11,16 In aged obese mice, Nrg4 intervention elevates p-AMPK levels in the liver, reduces p-mTOR levels, activates autophagy, and thereby alleviates hepatic steatosis.27 In vitro experiments demonstrate that Nrg4 also stimulates autophagy in L-02 cells and reduces lipid accumulation by activating the AMPK/mTOR signaling pathway.11 Activation of the MAPK signaling pathway depends on Shc. All ErbB4 variants contain Shc-binding sites in the cytoplasmic structure, which can associate with Shc, thereby activating the MAPK (Erk-1/2) signaling pathway, participating in the regulation of processes such as cell proliferation, differentiation, and metabolism. The STAT5 signaling pathway plays a crucial role in regulating Nrg4-mediated lipid metabolism. Nrg4 significantly promotes STAT5 phosphorylation, thereby inhibiting the transcriptional activity of liver X receptor (LXR). This leads to reduced expression of steroid-regulator element-binding factor 1 (Srebf1), acetyl-CoA carboxylase (Acaca), stearoyl-CoA desaturase 1 (SCD1), and fatty acid synthase (FASN).11 The STAT5 signaling pathway also modulates inflammatory responses and protects against glucose transport, thereby contributing to Nrg4’s improvement of diet-induced insulin resistance.1,3,5

Characteristics and Mechanisms of Insulin Resistance

Insulin resistance (IR) refers to the pathological state where peripheral target organs such as the liver, adipose tissue, and skeletal muscle exhibit decreased sensitivity to insulin. This manifests as the inability of insulin to effectively promote glucose uptake, utilization, and storage. To maintain blood glucose homeostasis, the body compensatorily secretes excessive insulin, leading to hyperinsulinemia and subsequently elevated blood glucose levels.28,29 As the core pathophysiological basis for metabolic disorders, insulin resistance is extensively implicated in the development and progression of type 2 diabetes, metabolic syndrome, obesity, and non-alcoholic fatty liver disease.28,30,31 It is frequently accompanied by pathological alterations such as chronic inflammation,32 dysregulated autophagy,33 and mitochondrial dysfunction,31 which intertwine and synergistically promote IR progression. Additionally, IR is modulated by multiple factors, including abnormal adipokine secretion, gut microbiota dysbiosis, and genetic variations (Figure 1).30

Figure 1 A schematic diagram of the core pathological mechanisms of insulin resistance (IR). The diagram illustrates three interconnected signaling pathways driving IR development: long-term fat accumulation recruits a large number of macrophages to infiltrate, leading to the release of pro-inflammatory cytokines, which impairs insulin signaling and GLUT4 transport. Obesity/high-fat diet-induced mitochondrial dysfunction disrupts insulin signaling through oxidative stress and metabolic defects. Dysregulation of autophagy not only leads to lipid accumulation, promoting abnormal activation of inflammatory pathways and pancreatic β-cell dysfunction, but also results in mitochondrial clearance defects, exacerbating oxidative stress and IR. These pathways synergistically form the pathological network of IR in metabolic syndrome.

Abbreviations: GLUT4, Glucose transporter type 4; TNFα, Tumor necrosis factor α; IL, 6-Interleukin-6.

Chronic inflammation is a hallmark of insulin resistance, characterized by infiltration of inflammatory cells into local tissues and persistent low-level release of pro-inflammatory factors, which can cause long-term damage to the insulin signaling pathway. Lipotoxicity is a key trigger for this inflammatory response. Adipose tissue is not merely a storage organ but also a crucial immune regulatory organ. When fat is excessively accumulated, adipocytes rupture and die, disrupting the local microenvironment and recruiting large numbers of macrophages, forming an inflammatory cycle composed of adipocytes and macrophages.34,35 Monocytes in adipose tissue differentiate into macrophages, forming what are known as adipose tissue macrophages (ATMs).35,36 Following infiltration, macrophages can polarize into pro-inflammatory M1 macrophages, which release pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNFα), interleukin-6 (IL-6), and interleukin-1β (IL-1β), thereby inhibiting insulin signaling.37,38 TNFα can also directly bind to the insulin receptor (InsR) or activate the mitogen-activated protein kinase (MAPK) pathway, phosphorylating serine/threonine residues of insulin receptor substrate (IRS). This prevents IRS binding to InsR and tyrosine phosphorylation, thereby disrupting downstream insulin signaling.38,39 IL-6 can inhibit GLUT4 gene expression and membrane transport by activating the transcription factor NF-κB, thereby reducing the glucose uptake capacity of skeletal muscle and adipocytes.38,40 Additionally, adipose tissue secretes adipokines such as adiponectin and resistin. Additionally, adipose tissue can also secrete adipokines such as adiponectin and resistin. Among these, resistin can promote macrophage activation and the secretion of pro-inflammatory factors, further exacerbating the inflammatory response. Conversely, a reduction in adiponectin levels due to lipid accumulation would weaken its anti-inflammatory and insulin-sensitizing effects,38,40 thereby forming a vicious circle of inflammation and IR.

Autophagy is a conserved process by which cells degrade damaged organelles, abnormal proteins, and excess lipids through lysosomes, playing a crucial role in maintaining intracellular homeostasis and regulating energy metabolism.41,42 Under normal physiological conditions, autophagy can maintain insulin sensitivity by clearing damaged mitochondria, degrading excessive lipids, reducing oxidative stress, and modulating inflammatory responses.43–45 In the state of IR, autophagy function is abnormally downregulated or dysfunctional, becoming a significant pathogenic mechanism that exacerbates IR, and the manifestations and impacts of autophagy dysregulation vary across tissues.46 In adipose tissue, impaired autophagy can lead to lipid metabolism disorders, with a large amount of triglycerides accumulating in adipocytes, causing adipocyte hypertrophy and abnormal function and promoting the secretion of pro-inflammatory factors.47,48 Saturated fatty acids such as palmitic acid can block the autophagy stream by suppressing the expression of autophagy-related genes (Atg5, Atg7), leading to intracellular lipid deposition. This subsequently activates the NF-κB inflammatory pathway, exacerbating adipose tissue inflammation and insulin resistance (IR).49,50 In skeletal muscle and liver, autophagy dysregulation leads to the accumulation of damaged organelles, particularly damaged mitochondria, thereby triggering increased oxidative stress and further injury to the insulin signaling pathway.51 Furthermore, dysregulation of autophagy in pancreatic β-cells also plays an important role in the progression of IR. Palmitate can lead to the accumulation of intracellular misfolded proteins and damaged mitochondria by inhibiting β-cell autophagy, thereby inducing lipotoxicity and oxidative stress,52,53 which eventually leads to apoptosis and dysfunction of β cells, making insulin secretion insufficient, unable to compensate for peripheral IR, and accelerating blood sugar rise.54,55

Mitochondria, as the core cellular organelles of energy metabolism, generate ATP through oxidative phosphorylation while participating in processes such as lipid metabolism, ROS production, and clearance. Their structural and functional abnormalities are significant pathological foundations for the occurrence of IR, particularly playing a critical role in obesity- and high-fat diet-induced IR. Factors such as obesity, high-fat diets, and hyperglycemia can lead to reduced mitochondrial numbers, structural abnormalities, and impaired function in target organs, including adipocytes, skeletal muscle cells, and hepatocytes.56–59 This, in turn, induces IR through multiple pathways. Mitochondrial dysfunction first leads to insufficient energy generation, thereby disrupting normal glucose and lipid metabolism.60 Skeletal muscle is the main organ for glucose uptake and utilization, and when its mitochondrial function is impaired, oxidative phosphorylation efficiency decreases, ATP production decreases, and cells compensate by increasing glycolysis to maintain energy supply, resulting in lactic acid accumulation and glucose utilization disorders. Mitochondrial lipid oxidative capacity decreases, leading to lipid accumulation in skeletal muscle and liver, inducing lipotoxicity that further damages the insulin signaling pathway.61,62 Mitochondrial dysfunction can also lead to excessive ROS generation, triggering oxidative stress.63 Under normal conditions, ROS generated by mitochondria can be cleared by the body’s antioxidant system. When mitochondrial structure is damaged, ROS production exceeds antioxidant capacity, and excessive ROS oxidatively damages insulin signaling molecules such as InsR and IRS, thereby inhibiting InsR tyrosine phosphorylation and IRS activation and blocking insulin signal transduction.64–66

Notably, autophagy is closely linked to inflammation and mitochondrial function. Impaired autophagy can exacerbate mitochondrial dysfunction, thereby promoting ROS production and the release of inflammatory mediators. Conversely, these inflammatory mediators further suppress autophagy activity, creating a cascading amplification effect that collectively drives the progression of insulin resistance (IR). Conversely, activating autophagy through pharmacological or genetic means can effectively clear excessive intracellular lipids and damaged organelles, reduce inflammatory responses and oxidative stress, improve insulin sensitivity, and provide potential targets for intervention in IR.

Nrg4 and Insulin Resistance Regulation of Glucose Metabolism by Nrg4

Nrg4 regulates glucose metabolic homeostasis in a multi-signaling-pathway crosstalk mode, with its core function relying on the synergistic action of three major mechanisms: inhibition of inflammation, regulation of autophagy, and protection of mitochondrial function. It achieves this by modulating glucose transport, hepatic gluconeogenesis, and insulin signal integrity, ultimately enhancing insulin sensitivity and lowering blood glucose levels (Figure 2).

Figure 2 A schematic diagram of glucose metabolic homeostasis mediated by Nrg4. After Nrg4 binds to its receptor ErbB4, it activates the PI3K/Akt and AMPK/mTOR signaling pathways and improves mitochondrial integrity. Upon activation of the PI3K/Akt pathway, it not only protects GLUT4 storage vesicles to ensure glucose uptake but also inhibits inflammatory pathways, reducing the release of inflammatory cytokines. Activation of the AMPK/mTOR pathway can activate autophagy to reduce lipotoxicity and protect β-cell function. Improving mitochondrial integrity reduces reactive oxygen species such as H2O2, alleviating oxidative stress. NRG4 maintains blood glucose balance and improves insulin resistance through these multiple pathways.

Abbreviations: AKT, serine/threonine kinase; PI3K, Phosphoinositide 3‑kinase; AMPK, AMP-activated protein kinase; mTOR, Mechanistic target of rapamycin; GLUT4, Glucose transporter type 4; Bcl-2, B-cell lymphoma 2; MFN2, Mitofusin 2; TIM44, Translocase of inner mitochondrial membrane 44.

Nrg4 regulates GLUT4 function, a key glucose transporter, by modulating the autophagy pathway. In the basal metabolism of adipocytes, Nrg4 binds to the ErbB4 receptor, activates the PI3K/Akt pathway, and maintains the activity of the mammalian target of rapamycin complex 1 (mTORC1).67 mTORc1 acts as a negative regulator of intracellular autophagy. When sufficient Nrg4 is expressed, it suppresses autophagic flux, preventing excessive degradation of cellular structures and proteins.67 Research shows that in Nrg4-deficient cells, the autophagy marker LC3-II is significantly elevated, accompanied by changes in autophagy substrates (such as p62), indicating enhanced autophagy activity.67,68 Under normal insulin stimulation, GLUT4 rapidly translocates from intracellular storage vesicles (GSVs) to the cell membrane, allowing glucose to enter cells for metabolism.26,69 However, Nrg4 knockdown in adipocytes is associated with increased autophagic flux, accelerated degradation of GLUT4 storage vesicles, reduced GLUT4 protein levels, and impaired glucose uptake,67 suggesting that Nrg4 may protect GSVs from autophagic turnover. Whether this mechanism operates in human insulin resistance requires further investigation. Research has demonstrated that treating Nrg4-knockdown adipocytes with the lysosomal inhibitor bafilomycin A1 (which blocks autophagy flux) effectively restores GLUT4, IRAP, Syntaxin-6, and TBC1D4 levels in Nrg4 KD cells to control levels.68 This finding further confirms that the mechanism by which Nrg4 protects GLUT4 is achieved through inhibition of the autophagy pathway. A high-fat environment inhibits the autophagy capacity of β-cells, leading to cell death. Supplementing with Nrg4 can reactivate the autophagy pathway, promoting the clearance of damaged organelles and the metabolism of excessive fatty acid byproducts, thereby ameliorating lipotoxicity in pancreatic β-cells.70 Following autophagy activation, Nrg4 upregulates the anti-apoptotic protein Bcl-2 while downregulating pro-apoptotic proteins Bax and caspase-3, ultimately safeguarding β-cell function.70 Thus, based on evidence from different experimental models, Nrg4 has been reported to either suppress or activate autophagy depending on cellular context and metabolic stress. In healthy adipocytes, Nrg4 sustains mTORC1 activity, limiting autophagic flux and preserving GLUT4 storage vesicles. In contrast, under lipotoxic conditions (eg, high‑fat diet), Nrg4 activates AMPK/mTOR signaling to enhance autophagic clearance of lipid droplets and damaged organelles.71,72 Whether Nrg4 directly “switches” the direction of autophagy within the same cell type under different conditions remains to be established. The current data suggest context-dependent effects rather than a true bidirectional regulatory switch.

Chronic inflammation is a major contributor to insulin resistance, and Nrg4 exerts significant anti-inflammatory effects, thereby improving insulin resistance. The presence of Nrg4 suppresses NF-κB transcriptional activity, reducing its activation of pro-inflammatory gene promoters.23 Current evidence indicates that this effect is indirect, mediated primarily through ErbB4-dependent activation of Akt, which in turn phosphorylates and stabilizes IκBα, thereby retaining NF-κB in the cytoplasm and preventing its nuclear translocation. Direct binding or direct inhibition of NF-κB by Nrg4 has not been demonstrated. Direct binding or direct inhibition of NF-κB by Nrg4 has not been demonstrated.3,23 This consequently reduces the expression of key pro-inflammatory factors such as TNF-α, IL-1β, IL-6, and IFN-β. Multiple studies indicate that knockdown (KD) of Nrg4 activates the NF-κB pathway in 3T3-L1 adipocytes, leading to upregulation of pro-inflammatory cytokines (such as TNFα, IL-1β, IL-6, and IFN-β).67,72 Supplementation with Nrg4 reverses these inflammatory responses. Nrg4-mediated suppression of the NF-κB pathway synergizes with its regulation of autophagy to dampen inflammatory responses, which may help preserve insulin receptor expression and protect GLUT4 storage vesicles from excessive autophagic degradation.11,67 These findings suggest a protective role for Nrg4 in maintaining insulin signaling integrity, although direct evidence in human tissues remains limited. Nrg4-mediated activation of ErbB4 can transactivate the PI3K/Akt pathway, thereby enhancing insulin signal transduction in an insulin-independent manner. This effect is indirect and occurs via ErbB4 receptor tyrosine kinase activity, rather than through direct interaction with insulin receptor or insulin receptor substrates. Furthermore, it enhances insulin sensitivity by inhibiting inhibitory factors in the insulin signaling pathway through phosphorylation (eg, Ser phosphorylation of IRS-1).3,11 Beyond the aforementioned pathways, Nrg4 inhibits the polarization of macrophages toward a pro-inflammatory phenotype (M1 type),73,74 reduces the expression of the macrophage marker gene monocyte chemotactic protein-1 (MCP-1), increases the expression of the M2 macrophage marker gene CD163, and improves inflammatory responses.1,75 In transgenic mice or animal models treated with Nrg4 gene transfer, it reduces mRNA levels of inflammatory genes in epididymal white adipose tissue (WAT), demonstrates improved glucose tolerance test (GTT) results, decreased insulin levels, and enhanced insulin signaling pathways in peripheral tissues, further improving insulin sensitivity.76,77 Research indicates that defects in the Nrg4-ErbB4 signaling pathway reduce adipose tissue angiogenesis, elevate inflammation levels, and exacerbate insulin resistance. In contrast, Nrg4 transgenic mice can compensatorily promote adipose tissue angiogenesis, thereby improving insulin resistance.74,77

Mitochondrial function protection provides the energy metabolic foundation for glucose homeostasis. Nrg4 maintains normal expression of mitochondrial fusion protein (MFN2) and mitochondrial inner membrane protein transport complex subunit (TIM44), thereby improving mitochondrial morphology and structural integrity.73 Simultaneously, it reduces the production of reactive oxygen species (ROS), such as H2O2, and suppresses oxidative stress.73 Reactive oxygen species (ROS) are highly reactive molecules that, when excessive, damage intracellular proteins, lipids, and DNA while disrupting insulin signaling pathways.78,79 Inhibiting oxidative stress involves eliminating these harmful free radicals to prevent cellular structural damage, protect organelle function, and thereby support normal insulin function.80,81 Research indicates that treating Nrg4-knockdown adipocytes with the antioxidant N-acetylcysteine reverses oxidative stress, thereby reducing TNFα gene expression. This partially restores insulin receptor and GLUT4 expression, safeguards glucose transport efficiency, and enhances insulin sensitivity.67,73 Simultaneously, Nrg4 induces the expression of mitochondrial respiratory chain-related genes, enhancing mitochondrial electron transport capacity. This increases mitochondrial oxygen consumption and glucose oxidation rates, promoting the shift of glucose from storage to utilization and thereby lowering blood glucose levels.5 Research shows that Nrg4 transgenic mice exhibit higher oxygen consumption rates (VO2) and energy expenditure in metabolic cage experiments,5 which is consistent with increased overall metabolic activity. However, these whole-body measurements do not directly prove enhanced mitochondrial function in specific tissues; they could also reflect increased physical activity or thermogenesis unrelated to mitochondrial coupling efficiency. Furthermore, the N-terminal EGFL peptide fragment released by Nrg4 crosses the blood-brain barrier to specifically bind to hypothalamic ErbB4 receptors, activating oxytocin neurons and promoting oxytocin release.76 This process directly stimulates insulin secretion, a mechanism also dependent on mitochondrial-mediated energy supply. Research has shown that central administration of recombinant Nrg4 (rNrg4) improves glucose tolerance and reduces blood glucose levels in diet-induced obese mice.76 These findings raise the possibility of a positive feedback loop in which Nrg4 enhances peripheral insulin sensitivity and, via central actions, promotes insulin secretion. However, direct evidence for such a feedback loop in vivo is currently lacking, and the relative contribution of central versus peripheral Nrg4 signaling remains to be quantified.

Stable c-FLIPL and enhanced autophagy activity jointly reduce inflammation and improve insulin signaling pathways, thereby enhancing insulin sensitivity.3 The improved inflammatory microenvironment further ensures normal autophagy regulation and stable GLUT4 function, forming a synergistic regulatory chain of “anti-inflammation-autophagy-glucose transport”.

Regulation of Lipid Metabolism by Nrg4

A major consequence of Nrg4 signaling in lipid metabolism is the reduction of lipid accumulation and improvement of hepatic steatosis, with its primary mechanism involving synergistic effects such as inflammation suppression, autophagy activation, and mitochondrial function enhancement, thereby achieving homeostatic regulation across three dimensions: lipid synthesis inhibition, fatty acid oxidation promotion, and lipid transport metabolism (Figure 3).

Figure 3 Schematic diagram of lipid metabolism regulation mediated by Nrg4. After Nrg4 binds to its receptor ErbB4, it activates PI3K/Akt, upregulating PPARα/CPT1α to promote fatty acid β-oxidation and lipolysis. NRG4 also inhibits SREBP1c expression through the LXR-NF-κB-STAT5 axis and suppresses the activation of inflammatory pathways, improving the inflammatory microenvironment to inhibit lipid synthesis and reduce hepatic fat accumulation. Additionally, NRG4 induces autophagic lipid clearance and white adipose tissue browning while promoting the expression of thermogenesis genes in brown adipose tissue to increase energy expenditure, collectively improving lipid accumulation.

Abbreviations: STAT5, Signal transducer and activator of transcription 5; LXR, Liver X-activated receptor; PPARα, Peroxisome Proliferator-Activated Receptor α; CPT1α, Carnitine palmitoyltransferase 1α; SREBP1c, Sterol Reg-Ulatory Element Binding Protein-1c; AKT, Protein kinase B; PI3K, Phosphoinositide 3‑kinase; Ucp1, Uncoupling protein 1; Ucp3, Uncoupling protein 3; Cidea, Cell Death Inducing DFFA Like Effector A; Dio2, Iodothyronine deiodinase 2.

At the level of lipid synthesis inhibition, Nrg4 exerts its effects through the STAT5-LXR signaling pathway. Nrg4 binds to the ErbB receptor on the surface of hepatocytes, leading to the phosphorylation of signal transducer and activator of transcription 5 (STAT5).3,11 The activated STAT5 then inhibits the transcriptional activity of the liver X receptor (LXR) via a transrepressive mechanism.3 LXR, as an upstream regulator of SREBP1c—the key transcription factor activating lipid synthesis—thereby downregulates the expression of lipid synthesis-related genes (Srebf1, Acaca, Scd1, Fasn), directly reducing hepatic lipid synthesis.3,11 The inhibition of lipid biogenesis does not occur in isolation; the aforementioned process synergizes with the suppression of inflammation. Inflammatory factors typically interfere with the STAT5-LXR pathway, weakening the lipolytic effect of Nrg4.3 Nrg4 significantly reduces inflammatory cytokine levels in metabolic tissues by inhibiting the NF-κB signaling pathway.1 By improving the inflammatory microenvironment, Nrg4 may help preserve STAT5-LXR pathway activity, which could contribute to its lipidsuppressing efficacy and inhibition of lipid biogenesis.3 Direct evidence that Nrg4 maintains this pathway in an “unimpeded” state is still limited.

The core role of autophagy regulation in lipid metabolism is to promote lipid clearance and reduce lipid accumulation.82 In the liver, Nrg4 activates the AMPK pathway by binding to the ErbB4 receptor, directly phosphorylating the pre-SREBP1c precursor protein, thereby inhibiting the activation of sterol regulatory element-binding protein 1c (SREBP1c). SREBP1c is a key transcription factor regulating adipogenesis. By inhibiting SREBP1c, Nrg4 reduces the formation and accumulation of lipid droplets.27,83,84 Studies indicate that supplementing with exogenous Nrg4 or upregulating endogenous Nrg4 levels in non-alcoholic fatty liver disease (NAFLD) models significantly improves hepatic steatosis.11,16 Furthermore, Nrg4 activation of autophagy also helps clear excessive lipid droplets by upregulating autophagy-related proteins (such as LC3B-II) and downregulating p62, thereby maintaining lipid metabolic balance.16 Inflammation suppression creates favorable conditions for autophagy activation. Reducing inflammatory factors prevents abnormal regulation of the autophagy pathway, ensuring Nrg4’s positive induction of autophagy. Following autophagy activation, decreased lipid accumulation in turn reduces inflammatory factor production, forming a positive feedback loop of anti-inflammation, autophagy, and lipid clearance.23,85 Nrg4 stimulates white adipose tissue browning, enhances brown adipose tissue activity, promotes adipose tissue angiogenesis, and improves adipose tissue hypoxia and inflammation, thereby reducing lipid accumulation.76,86,87 Studies reveal that in mice overexpressing Nrg4, a high-fat diet leads to significantly increased browning of white adipose tissue, reduced adipocyte deposition, and decreased serum and hepatic triglyceride (TG) and total cholesterol (TC) levels.76 Nrg4 also stimulates energy expenditure and reduces lipid accumulation by activating the thermogenic function of brown adipose tissue (BAT). Gene transfer of Nrg4 enhances the expression of thermogenic genes in BAT, including Ucp1, Ucp3, Cidea, and Dio2.11,12 Enhanced mitochondrial function is a key driver in promoting fatty acid oxidation and reducing lipid accumulation. Nrg4 activates the PI3K/Akt signaling pathway by binding its receptor ErbB4.1 This pathway upregulates the expression of peroxisome proliferator-activated receptor alpha (PPARα) and its target gene, carnitine palmitoyltransferase 1a (CPT1a).1,73 As the gating enzyme for fatty acyl-CoA transport into mitochondria, increased CPT1a activity directly accelerates fatty acid uptake into mitochondria. This facilitates efficient β-oxidation, thereby alleviating lipid accumulation.88

Nrg4 regulates metabolic homeostasis through a finely tuned signaling network. It enhances glucose metabolism efficiency and improves insulin resistance by suppressing inflammation, maintaining autophagy balance, and protecting mitochondria. Meanwhile, in lipid metabolism, it comprehensively prevents lipid accumulation and hepatic steatosis by inhibiting genes involved in lipid synthesis, activating autophagy to clear lipid droplets, and enhancing mitochondrial fatty acid oxidation. These mechanisms collectively constitute Nrg4’s core role in metabolism.

Relevant Findings of Nrg4 in Clinical Research

Clinical studies investigating the association between Nrg4 and insulin resistance have produced a body of evidence that is substantial yet increasingly controversial. While preclinical studies consistently demonstrate an insulin-sensitizing effect of Nrg4, human studies have yielded conflicting results, with some reporting protective associations while others suggest neutral or even detrimental relationships. This section critically evaluates the existing clinical evidence, identifies methodological sources of inconsistency, and provides a systematic comparison of key studies.

Overview of Clinical Evidence

Several systematic reviews and meta-analyses have attempted to synthesize the growing body of clinical literature on Nrg4. A systematic review by Tutunchi et al concluded that Nrg4 expression is substantially downregulated in human obesity and that Nrg4 may contribute to the prevention of obesity-related metabolic complications by promoting brown adipose tissue activity and improving glucose homeostasis.89 More recently, Ziqubu et al conducted a systematic review encompassing obesity, gestational diabetes mellitus (GDM), T2DM, NAFLD, and cardiovascular diseases, concluding that ample clinical evidence supports reduced circulating Nrg4 levels in morbid obesity, inversely proportional to indices of metabolic syndrome including BMI, waist circumference, triglycerides, fasting plasma glucose, HOMA-IR, and hs-CRP.90 A meta-analysis by Tapak et al, pooling five case-control studies with 323 NAFLD cases and 308 controls, reported a pooled odds ratio of 0.72 (95% CI: 0.67–0.77), suggesting a protective association between serum Nrg4 levels and incident NAFLD.91 However, pooled observational biomarker studies remain vulnerable to confounding, and the meta-analysis itself calls for larger, more accurate study designs to corroborate findings. Additionally, a meta-analysis of GDM by Cindoglu et al including seven studies with 347 GDM cases and 362 controls found no significant difference in circulating Nrg4 levels between GDM and non-GDM groups in the primary analysis (SMD = −0.35, 95% CI: −1.17 to 0.48, p = 0.41, I2 = 96%), though sensitivity analyses and subgroup analyses did report significantly reduced levels in certain contexts, highlighting the methodological heterogeneity across studies.92

Key Clinical Studies

Individual clinical studies have reported markedly divergent findings. Kang et al first reported that serum Nrg4 levels were significantly higher in patients with newly diagnosed T2DM compared with controls without diabetes and were correlated with serum glucose levels and HOMA-IR.4 By contrast, Akshay et al studied 100 newly diagnosed T2DM patients and 100 matched controls, finding plasma Nrg4 levels significantly lower in diabetics (7949.76 ± 949.76 pg/mL vs 9143 ± 949.76 pg/mL, p < 0.0001), with significant negative correlations with fasting blood sugar (r = −0.303), postprandial blood sugar (r = −0.416), HbA1c (r = −0.433), and HOMA-IR (r = −0.514), alongside a positive correlation with HDL (r = 0.216).6 Similarly, Yan et al reported that circulating Nrg4 levels were significantly lower in newly diagnosed T2DM patients without peripheral neuropathy than in healthy controls, and further decreased in those with diabetic peripheral neuropathy (DPN), with a cutoff value of 1.58 ng/mL predicting DPN with 90.91% sensitivity and 54.55% specificity.93

In a study that contradicts the prevailing protective paradigm, Martínez et al found that serum Nrg4 levels were negatively correlated with insulin sensitivity (assessed by HOMA-IR and clamp-derived measures) and positively correlated with hs-CRP levels. Furthermore, they observed that Nrg4 attenuated mitochondrial respiration in human HepG2 cells without affecting expression of genes involved in lipid metabolism, suggesting potentially divergent effects in different species or experimental systems.7

For GDM, Al-Bayati and Saleh reported that circulating Nrg4 levels were reduced in GDM patients compared with healthy pregnant women, with a negative correlation with HOMA-IR. More recently, a matched case-control study of Chinese pregnant women found that elevated serum NRG4 levels were associated with lower GDM risk, with an optimal cutoff value of 0.58 ng/mL predicting GDM with 92% sensitivity and 42.5% specificity (AUC = 0.714).94 Cindoglu et al similarly reported significantly lower serum Nrg4 levels in the diabetes group versus controls (3.22 ± 2.16 vs 4.55 ± 0.96, p < 0.001).95

Sources of Clinical Controversy Issue 1: Circulating Nrg4 versus Tissue Expression

A fundamental issue confounding the interpretation of clinical studies is that human investigations overwhelmingly measure circulating Nrg4 levels, whereas the key mechanistic evidence from preclinical models concerns adipose tissue expression, hepatocyte signaling, or recombinant/gene-transfer interventions. Circulating Nrg4 levels reflect systemic secretion, clearance, and potential cross-tissue contributions, whereas tissue expression captures local autocrine/paracrine effects. Wang et al originally demonstrated that adipose tissue Nrg4 expression, not circulating levels, was reduced in rodent and human obesity and that transgenic expression in adipose tissue was sufficient to attenuate hepatic lipogenesis.3 A first expression study on human liver samples by Bograya et al found decreased ERBB4 and NRG4 mRNA expression in livers of subjects with high BMI but not in those with NAFLD, revealing new research directions and underscoring that tissue-specific expression patterns may not correlate with circulating levels.96 Thus, the lack of standardized, paired measurements of both circulating and tissue-specific Nrg4 expression in human studies represents a major translational gap.

Issue 2: Cross-Sectional Design and Causal Inference

The vast majority of clinical studies examining Nrg4 in relation to insulin resistance are cross-sectional in design, which cannot establish causality. Lower circulating Nrg4 levels in T2DM or NAFLD patients could be causal (ie, Nrg4 deficiency contributes to disease pathogenesis), compensatory (ie, the disease state triggers Nrg4 upregulation as a protective response), secondary to adipose tissue dysfunction (ie, obesity-induced inflammation downregulates Nrg4 expression), or purely assay-dependent.90 Dan et al comprehensively reviewed the relationship between circulating Nrg4 levels and T2DM-related vascular complications, noting that while Nrg4 is significantly decreased in adipose tissue and serum during aging, prospective cohort studies are warranted to confirm outcomes.1 Without longitudinal data or Mendelian randomization studies, the direction of causality remains unknown.

Issue 3: Heterogeneity of Insulin Sensitivity Endpoints

Clinical studies employ widely differing metrics for assessing insulin sensitivity, and these are not equivalent. HOMA-IR, which estimates insulin resistance from fasting glucose and insulin, is the most commonly used method due to its convenience but is heavily influenced by hepatic insulin resistance and has limited sensitivity for detecting changes in peripheral insulin sensitivity.6 Fasting insulin alone is sometimes used but is even less informative. OGTT-derived indices (eg, Matsuda index, ISI composite) provide a more integrated assessment of whole-body insulin sensitivity but are less commonly employed.97 The hyperinsulinaemic-euglycaemic clamp remains the gold standard but is rarely used in large-scale studies due to its complexity. Akshay et al stands out as one of the few studies that employed clamp-derived measures alongside HOMA-IR, enabling more robust comparisons of Nrg4 levels with both surrogate and direct measures of insulin sensitivity.6 This heterogeneity likely contributes substantially to the divergent findings across studies, as different metrics capture different aspects of insulin resistance (hepatic vs peripheral, fasting vs postprandial).

Issue 4: Inconsistency in NAFLD Findings

Although the meta-analysis by Tapak et al suggests a protective association between serum Nrg4 and NAFLD, individual studies have produced inconsistent results.91 Wang et al initially reported that human adipose tissue Nrg4 levels were related to body fat and liver fat mass.3 However, De Munck et al concluded that their study did not support a role for circulating Nrg4 in human NAFLD after rigorous adjustment for confounding variables.98 Bograya et al found decreased ERBB4 and NRG4 mRNA expression in livers of subjects with high BMI but not in those with biopsy-proven NAFLD, suggesting that NAFLD pathophysiology may involve alterations in Nrg4 signaling at the receptor level rather than changes in circulating Nrg4 per se.96 This further supports the notion that tissue-specific expression patterns may not correlate with circulating levels and that reliance on circulating Nrg4 as a surrogate for tissue function may be misleading.

Systematic Comparison of Clinical Studies

Table 1 summarizes the design, population, sample size, assay method, specimen type, insulin resistance metric, direction of association, adjustment variables, and key limitations of the main clinical studies discussed above. As shown in the table, all available studies are cross-sectional or case-control in design, with sample sizes ranging from 34 to 200 participants per study. Nearly all relied on circulating Nrg4 measurements (serum or plasma) and HOMA-IR as the primary insulin resistance metric, whereas only one study employed hyperinsulinaemic-euglycaemic clamp-derived indices. Adjustment for confounders varied considerably across studies.

Table 1 Summary of Clinical Studies Investigating Nrg4 in Relation to Insulin Resistance

Therapeutic Potential and Strategies for Nrg4 Recombinant Nrg4 Protein Therapy

Recombinant Nrg4 protein has shown promising therapeutic effects in animal studies. Recombinant human Nrg4-Fc (hNrg4-Fc) fusion protein has a prolonged plasma half-life, and the effective concentration can be maintained in the body for 3–5 days after a single injection,12,99 thereby it can effectively stimulate the ERBB4 signal conduction. This kind of protein can specifically activate the ERBB4 receptor on the surface of the target cell, initiate the downstream signal cascading reaction, regulate the lipid metabolism homeosis, thus improving the efficiency of insulin signal transduction and alleviating metabolic disorders from the source. Through fixed mutation, glycosylation modification and other means, the stability, targeting and biological activity of recombinant proteins can be further enhanced and immunogenicity can be reduced. In clinical application, this strategy is expected to treat obesity-related metabolic diseases, non-alcoholic fatty liver and other diseases through intravenous or subcutaneous injection to meet the treatment needs of different patients.3,12

Gene Therapy

Gene therapy achieves a long-term and stable increase in Nrg4 levels through exogenous introduction of Nrg4 genes or regulation of endogenous Nrg4 expression, which is a long-term strategy for the treatment of chronic metabolic diseases. The strategy can be implemented through various gene transfer systems, including viral and non-viral vectors. Different vectors have different advantages, and appropriate methods can be selected according to the purpose of treatment. For example, virus vectors have high transduction efficiency and continuous expression, which is suitable for long-term intervention;75 Non-viral vectors have high safety and low immunogenicity, making them easier to convert into clinical applications. The core value of gene therapy lies in the multi-target regulation of the metabolic network, which not only enhances the expression of Nrg4, but also coordinates the regulation of fat factor secretion, inflammatory response and liver lipid metabolism, so as to improve metabolic disorders as a whole.75,100 However, therapeutic delivery via viral vectors remains in the validation stage, as it may still face significant safety issues and other challenges, indicating a longer development process ahead.

Cell Therapy

Cell therapy based on fatty mesenchymal stem cells (ADSC), combined with Nrg4 gene modification, has developed into a collaborative treatment model that combines the regeneration and repair ability of stem cells with the metabolic regulation of Nrg4. ADSCs (obtained from adipose tissue, thus ensuring these advantages) have the advantages of wide availability, easy access, low immunogenicity and multidirectional differentiation potential, making them ideal carriers for the Nrg4 gene. After engraftment in target organs, they continuously secrete Nrg4, achieving precise regulation of the local microenvironment. Nrg4-modified ADSCs (Nrg4-ADSCs) exert therapeutic effects through multiple mechanisms. On one hand, they secrete Nrg4 to activate the ERBB4 signal, improving insulin resistance and lipid metabolism; on the other hand, relying on the paracrine effects of ADSCs, they secrete cytokines, growth factors, and other substances, inhibiting inflammatory responses and promoting tissue repair, particularly demonstrating synergistic improvement effects on hepatic steatosis and adipose tissue inflammation.101,102

Beyond the aforementioned direct therapeutic strategies targeting Nrg4, metabolic regulation can be achieved by indirectly modulating Nrg4 expression or key molecules in its signaling pathway, offering additional approaches for disease treatment. In the field of drug development, targeted drug design can be focused on key molecules of the Nrg4 signaling pathway, including ERBB4 receptor agonists/modulators, AMPK activators, mTOR inhibitors, etc. ERBB4 agonists can mimic the action of Nrg4, thereby activating downstream signaling; AMPK activators can indirectly enhance Nrg4 expression by regulating energy metabolism pathways; mTOR inhibitors can inhibit lipid synthesis, thereby synergistically improving metabolic disorders in the presence of Nrg4. Furthermore, certain natural products have been shown to regulate Nrg4 expression, providing a direction for developing drugs from natural sources.5,103 These indirect strategies can be combined with direct therapeutic approaches to form multidimensional treatment regimens, enhancing therapeutic efficacy.

Safety and Potential Risks

As a potential therapeutic target for insulin resistance-related diseases, the core premise of clinical translation is to solve safety issues. Nrg4 functions through the ErbB4 receptor, but ErbB4 biology is extremely complex, tissue-specific, and stage-dependent. As a specific ligand for ErbB4, Nrg4 induces tyrosine phosphorylation of ErbB4, activating downstream MAPK/ERK and PI3K/Akt pathways to promote cell proliferation.88