Neuregulin-4 (NRG4): Brown Adipose Tissue-Derived Batokine Protecting Against NAFLD via ErbB3/ErbB4 Signaling in Research

# Neuregulin-4 (NRG4): Brown Adipose Tissue-Derived Batokine Protecting Against NAFLD via ErbB3/ErbB4 Signaling in Research

Neuregulin-4 is a member of the neuregulin (NRG) subfamily of EGF-like growth factors that has emerged as a key mediator of inter-organ communication between brown adipose tissue (BAT) and liver. Identified in 2014 as a BAT-enriched secreted factor that inhibits hepatic de novo lipogenesis, NRG4 exemplifies the growing concept of "batokines" — hormones secreted by BAT that coordinate energy homeostasis across distant organs. Its clinical relevance is underscored by consistent findings of reduced circulating NRG4 in obesity, NAFLD, type 2 diabetes, and metabolic syndrome, while animal models of NRG4 deficiency develop accelerated hepatic steatosis and steatohepatitis.

The Neuregulin Family and ErbB Receptor System

Neuregulins are a family of four related proteins (NRG1-4) encoded by distinct genes that share an EGF-like domain responsible for binding and activating ErbB receptor tyrosine kinases. The neuregulin family emerged from studies of developmental biology and cancer: NRG1 was first identified as heregulin, a ligand for ErbB2 (HER2), and NRG1 signaling through ErbB2/ErbB3 and ErbB2/ErbB4 heterodimers is essential for cardiac development, neural crest migration, and Schwann cell myelination.

The four human neuregulin genes differ substantially in their tissue expression, protein structure, and receptor specificity:

• •NRG1: Most complex locus, multiple isoforms via alternative splicing; activates ErbB2/ErbB3 and ErbB2/ErbB4; expressed broadly in heart, nervous system, skeletal muscle
• •NRG2: ErbB3/ErbB4 ligand; expressed in brain; less studied
• •NRG3: ErbB4 selective; expressed in brain and nervous system
• •NRG4: ErbB4 selective (and ErbB3 to a lesser extent); predominantly expressed in BAT and WAT

Discovery as a BAT-Derived Metabolic Regulator

1. BAT-enriched expression: NRG4 mRNA in mouse interscapular BAT was 5-10 fold higher than in WAT or liver

2. NRG4 secretion: BAT explants secreted NRG4 protein; plasma NRG4 was measurable and BAT-derived

3. Hepatic NRG4 receptor: The liver highly expresses ErbB4 (the primary NRG4 receptor), consistent with liver as a target

4. NRG4 inhibits hepatic lipogenesis: Recombinant NRG4 treatment of primary hepatocytes suppressed fatty acid synthesis, reducing expression of SREBP1c, FASN, ACC1, and SCD1

5. Nrg4−/− mice develop NAFLD: On normal chow, Nrg4-knockout mice showed mild steatosis; on high-fat diet (HFD), they developed severe hepatic steatosis, elevated ALT, and histological NASH compared to wild-type littermates

6. Overexpression prevents HFD steatosis: Adenoviral NRG4 overexpression in Nrg4−/− mice rescued hepatic steatosis, and liver-targeted NRG4 administration in diet-induced obese (DIO) mice reduced steatosis

7. Plasma NRG4 is reduced in obese mice and humans: Circulating NRG4 was inversely correlated with fat mass, liver steatosis score, and plasma lipids

This paper established NRG4 as the first identified BAT-to-liver endocrine axis and coined the term "batokine" for BAT-derived hormonal signals.

Gene and Protein Structure

NRG4 Gene

The human NRG4 gene is located on chromosome 15q23 and encodes a type I transmembrane protein of 115 amino acids (shorter than NRG1-3 which have much more complex multi-domain structures). The NRG4 protein structure is notably simpler than other neuregulins:

• •Signal peptide (residues 1-31): Directs to ER for processing
• •Spacer/stalk domain (residues 32-72): Linker region preceding the EGF domain
• •EGF-like domain (residues 73-113): The receptor-binding domain with six conserved cysteines forming three disulfide bonds; the minimal active fragment
• •Transmembrane domain (residues 114-133): Single-pass; suggests juxtacrine activity before shedding
• •Short cytoplasmic tail (residues 134-~140)

NRG4 is processed similarly to other membrane-anchored EGF family ligands: it is synthesized as a transmembrane precursor and then shed from the cell surface by metalloprotease-mediated ectodomain shedding (ADAM10 and ADAM17 have been implicated). The shed EGF-like domain (~5 kDa) is the soluble circulating form that signals in an endocrine fashion to the liver. The small size of the EGF-like domain and its simple structure are notable compared to the complex multi-domain architecture of NRG1.

The EGF-like domain binds ErbB4 with high affinity (Kd ~1-10 nM) and ErbB3 with lower affinity, primarily triggering ErbB4 homodimerization or ErbB4/ErbB3 heterodimerization.

Receptor Signaling: ErbB3 and ErbB4

ErbB4 (HER4) is a receptor tyrosine kinase of the ErbB/HER family:

• •Expressed abundantly in liver, brain (neurons), heart, kidney
• •Upon NRG4 binding, ErbB4 undergoes homodimerization or heterodimerization with ErbB3
• •Trans-autophosphorylation of multiple cytoplasmic tyrosine residues (Y1172, Y1248, Y1284)
• •Recruitment of adaptor proteins (Grb2, Shc, PI3K p85 subunit)
• •Activation of downstream: PI3K/Akt, ERK1/2 MAPK, STAT3, and in some contexts PLCγ

In hepatocytes specifically, the NRG4-ErbB4 signal converges on suppression of SREBP1c (sterol regulatory element-binding protein 1c), the master transcription factor for de novo lipogenesis:

1. NRG4 → ErbB4 → PI3K/Akt activation

2. Akt phosphorylates and suppresses the LXR (liver X receptor) coactivator function

3. Reduced LXR activity → reduced SREBP1c transcription → reduced FASN, ACC1, SCD1 expression

4. Net: suppressed de novo fatty acid synthesis

Additionally, NRG4-ErbB4-ERK1/2 signaling in hepatocytes activates AMPK in some models, contributing to hepatic fatty acid oxidation through ACC2 phosphorylation (reducing malonyl-CoA and de-repressing CPT1).

BAT Biology and the Batokine Concept

Brown adipose tissue (BAT) is unique in its ability to dissipate chemical energy as heat through uncoupled mitochondrial respiration (via uncoupling protein 1, UCP1). In rodents, BAT is a substantial endocrine organ, particularly when thermogenically activated by cold exposure or β3-adrenergic agonists. In humans, active BAT depots exist (primarily in the supraclavicular region) and can be detected by ¹⁸FDG-PET imaging during cold exposure.

The "batokine" concept proposes that BAT secretes factors that signal to other organs to coordinate systemic metabolism with thermogenic activity. NRG4 was the first batokine identified that specifically links BAT activity to hepatic lipid homeostasis. The physiological logic is compelling: when BAT is thermogenically active (cold exposure, exercise), it secretes NRG4 to suppress hepatic lipogenesis, redirecting metabolic substrates toward BAT oxidation rather than hepatic storage. This BAT→liver circuit provides a systemic metabolic "override" that favors fat burning over fat storage during energy-demanding conditions.

Cold activation of NRG4: Cold exposure (4°C for 4 hours) in mice significantly increases NRG4 mRNA in BAT and plasma NRG4 concentrations, confirming thermogenic activation as a physiological stimulus. β3-adrenergic receptor agonists (CL316,243) similarly upregulate BAT NRG4 expression.

Exercise as NRG4 inducer: Muscle contraction and exercise upregulate irisin and other myokines; BAT may additionally upregulate NRG4 during exercise-associated thermogenesis. The relative contribution of BAT vs. skeletal muscle NRG4 secretion during exercise is being studied.

Hepatic Biology and NAFLD Protection

NAFLD/NASH Disease Model

NAFLD affects approximately 25% of the global adult population and encompasses a spectrum from simple steatosis to NASH (with inflammation and ballooning), fibrosis, cirrhosis, and hepatocellular carcinoma. De novo lipogenesis (DNL) — conversion of carbohydrates to fatty acids in hepatocytes — is a major driver of liver fat accumulation in NAFLD, contributing 15-25% of hepatic triglycerides in NAFLD patients vs. <5% in healthy individuals.

NRG4-ErbB4 signaling suppresses the DNL pathway at multiple transcriptional levels:

• •SREBP1c suppression: Reduces transcription of all lipogenic genes simultaneously
• •LXR modulation: LXRα promotes SREBP1c transcription; NRG4-Akt-mediated LXR suppression reduces this
• •ChREBP modulation: In some studies, NRG4 also suppresses carbohydrate response element-binding protein (ChREBP), another lipogenic transcription factor active during glucose surplus

Consequently, Nrg4−/− mice on HFD develop a dramatically accelerated NAFLD phenotype: more fat accumulation, higher NASH activity scores (NAS), elevated transaminases, and more rapid progression to fibrosis. Conversely, hepatic ErbB4 overexpression in DIO mice partially corrects steatosis.

NRG4 and Hepatic Inflammation (NASH)

Beyond lipogenesis suppression, NRG4 has documented anti-inflammatory effects in the liver:

• •NRG4 treatment reduces LPS-induced IL-6, TNF-α, and CXCL10 production from Kupffer cells (hepatic macrophages)
• •NRG4 suppresses NF-κB pathway in hepatocytes and Kupffer cells
• •In the methionine-choline deficient (MCD) diet model of NASH, NRG4 administration reduces steatohepatitis severity
• •NRG4 reduces hepatic fibrosis markers (TGF-β, α-SMA, collagen I) in some fibrosis models, suggesting hepatic stellate cell modulation

Plasma NRG4 in Human Metabolic Disease

Clinical studies have consistently found plasma NRG4 reduced in metabolic disease:

• •Obesity: BMI inversely correlates with plasma NRG4; morbidly obese subjects have 40-60% lower NRG4 vs. lean controls
• •NAFLD: Plasma NRG4 inversely correlates with NAS score, liver fat fraction (by MRI-PDFF), and AST/ALT; significantly lower in NASH vs. simple steatosis
• •Type 2 Diabetes: Plasma NRG4 is lower in T2DM, inversely correlating with HbA1c, fasting insulin, and HOMA-IR
• •Metabolic Syndrome: Lower in subjects meeting MetS criteria, inversely correlating with triglycerides and fasting glucose
• •Bariatric surgery: Multiple studies show plasma NRG4 increases significantly after Roux-en-Y gastric bypass or sleeve gastrectomy, correlating with improvement in liver steatosis and insulin resistance

The reference range for plasma NRG4 in healthy adults is approximately 2-8 ng/mL (by ELISA), though values vary between studies. The reduction in metabolic disease presumably reflects BAT dysfunction (reduced thermogenic activity and secretory capacity in obese, insulin-resistant individuals), creating a vicious cycle: obese → less functional BAT → less NRG4 → more hepatic lipogenesis → more steatosis → worse metabolic disease.

Additional Biological Functions

Pancreatic islets: ErbB4 is expressed in pancreatic β-cells, and NRG1/NRG4 signaling through ErbB4 has been shown to promote β-cell survival and GSIS enhancement. NRG4 may therefore also act on the pancreas, though this is less well-characterized than its hepatic effects.

Cardiac biology: ErbB4 is critically important in cardiomyocytes (NRG1-ErbB2/ErbB4 axis is required for cardiac development; trastuzumab/Herceptin cardiotoxicity reflects ErbB2 blockade in cardiomyocytes). NRG4 may similarly have cardiac effects through ErbB4, though this has not been a primary focus.

BAT browning and thermogenesis: NRG4 may act in an autocrine/paracrine fashion within BAT itself. ErbB4 is expressed in brown adipocytes, and NRG4 treatment of brown adipocyte cell lines promotes UCP1 expression and mitochondrial biogenesis in some models — suggesting a positive feedback mechanism for maintaining thermogenic capacity.

Adipocyte differentiation: NRG4-ErbB4 signaling promotes brown/beige adipocyte differentiation from preadipocytes in some in vitro systems, potentially contributing to BAT mass maintenance.

Research Tools and Experimental Systems

Current Research Frontiers

NRG4 as NAFLD/NASH biomarker and treatment target: Given its consistent reduction in NAFLD and correlation with disease severity, plasma NRG4 is being evaluated as a non-invasive NAFLD biomarker and therapeutic target. Recombinant NRG4 or ErbB4-selective agonists could represent anti-steatotic agents — particularly attractive because they would target a BAT-liver crosstalk circuit rather than single-organ pathways.

Human BAT activity and NRG4: The relationship between human BAT activity (quantifiable by ¹⁸FDG-PET) and plasma NRG4 is being studied. If BAT activity determines NRG4 secretion in humans as in rodents, then interventions that reactivate human BAT (cold thermogenesis, β3-AR agonists in clinical development) could raise NRG4 and protect against NAFLD.

NRG4 in MASH clinical trials: The pipeline for metabolic-associated steatohepatitis (MASH, the new preferred terminology for NASH) includes multiple molecular targets. NRG4-ErbB4 pathway activation is being explored as a complementary approach to GLP-1 receptor agonists (which have shown efficacy in NASH).

ErbB4 as a cancer risk: ErbB4 is a validated oncogene in some cancers (notably breast cancer in combination with HER2). The use of NRG4-ErbB4 agonists for NAFLD treatment must carefully evaluate cancer-promoting potential, particularly in the context of pre-existing liver disease (cirrhosis, HCC risk).

Integration with exercise biology: Cold exposure, exercise, and β-adrenergic activation all increase BAT NRG4. Understanding how exercise-induced NRG4 contributes to the well-established hepatoprotective effects of physical activity on NAFLD is an active area.

Conclusion

Neuregulin-4 provides a compelling example of inter-organ endocrine communication where BAT acts as a source of hepatoprotective batokines. Its discovery resolved a long-standing question about why BAT-active states (cold adaptation, exercise) are associated with reduced hepatic lipid accumulation, and it positioned the BAT→liver NRG4-ErbB4 axis as a physiologically relevant circuit for coordinating fat storage with thermogenic demand. The consistent reduction of circulating NRG4 in obesity, NAFLD, and metabolic syndrome suggests a pathological loss of BAT-mediated hepatic protection — a concept with clear therapeutic implications. ErbB4-targeted agonism, or interventions that restore BAT function and NRG4 secretion, represent promising anti-NAFLD/NASH strategies currently under preclinical and early clinical exploration. Recombinant NRG4 EGF domain protein, Nrg4−/− mice, and adenoviral NRG4 overexpression systems are the primary research tools for mechanistic and translational investigation.

Key References

1. Wang GX, Zhao XY, Meng ZX, et al. The brown fat-enriched secreted factor Nrg4 preserves metabolic homeostasis through attenuation of hepatic lipogenesis. Nat Med. 2014;20(12):1436-1443. PMID: 25401691

3. Chen LH, Hu B, Qiu WJ, et al. Neuregulin-4 is negatively associated with hepatic steatosis as well as liver fibrosis in patients with non-alcoholic fatty liver disease. J Diabetes Complications. 2019;33(7):499-504. PMID: 30967270

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References

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This article is intended for Research Use Only (RUO). The information provided describes laboratory research findings and does not constitute medical advice. Neuregulin-4 and related ErbB4 ligands are research tools and investigational agents. All research applications must comply with applicable institutional, local, and national regulations.