Hepatic neuregulin 4 signaling defines an endocrine checkpoint for steatosis-to-NASH progression

Abstract

Nonalcoholic steatohepatitis (NASH) is characterized by progressive liver injury, inflammation, and fibrosis; however, the mechanisms that govern the transition from hepatic steatosis, which is relatively benign, to NASH remain poorly defined. Neuregulin 4 (Nrg4) is an adipose tissue-enriched endocrine factor that elicits beneficial metabolic effects in obesity. Here, we show that Nrg4 is a key component of an endocrine checkpoint that preserves hepatocyte health and counters diet-induced NASH in mice. Nrg4 deficiency accelerated liver injury, fibrosis, inflammation, and cell death in a mouse model of NASH. In contrast, transgenic expression of Nrg4 in adipose tissue alleviated diet-induced NASH. Nrg4 attenuated hepatocyte death in a cell-autonomous manner by blocking ubiquitination and proteasomal degradation of c-FLIPL, a negative regulator of cell death. Adeno-associated virus-mediated (AAV-mediated) rescue of hepatic c-FLIPL expression in Nrg4-deficent mice functionally restored the brake for steatosis to NASH transition. Thus, hepatic Nrg4 signaling serves as an endocrine checkpoint for steatosis-to-NASH progression by activating a cytoprotective pathway to counter stress-induced liver injury.

Keywords: Hepatology; Signal transduction.

Figure 1. Human and mouse NASH are linked to induction of apoptosis and necroptosis in the liver.

(A) Immunoblots of total liver lysates from normal individuals and NASH patients. (B) qPCR analysis of gene expression in normal (n = 7) and NASH (n = 7) human livers. Data represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001, 2-tailed unpaired Student’s t test. (C) H&E, sirius red, F4/80 immunofluorescence, and TUNEL staining of liver sections from male C57BL/6J mice fed chow or NASH diet. Scale bars: 100 μm. (D) Plasma ALT, AST, and HMGB1 levels in mice fed chow (n = 4) or NASH diet (n = 4). Data represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001, 2-tailed unpaired Student’s t test. (E) qPCR analysis of hepatic gene expression. Data represent mean ± SEM. **P < 0.01; ***P < 0.001, 2-tailed unpaired Student’s t test. (F) Immunoblots of total liver lysates from mice fed chow or NASH diet. (G) qPCR analysis of Nrg4 expression in eWAT and BAT. Data represent mean ± SEM. *P < 0.05, 2-tailed unpaired Student’s t test.

Figure 2. Nrg4 deficiency exacerbates diet-induced NASH in mice.

Control WT (n = 12, black) and Nrg4-KO (n = 12, gray) male mice were fed NASH diet for 20 weeks, starting at 3 months of age. (A) Plasma ALT, AST, and HMGB1 levels and liver TAG content. Data represent mean ± SEM. *P < 0.05; **P < 0.01, 2-tailed unpaired Student’s t test. (B) H&E, sirius red, F4/80 immunofluorescence, and TUNEL staining of liver sections. Scale bars: 100 μm. (C) Quantification of sirius red, F4/80, and TUNEL staining images and liver hydroxyproline content. Data represent mean ± SEM. **P < 0.01; ***P < 0.001, 2-tailed unpaired Student’s t test. (D) Immunoblots of total liver lysates from NASH diet–fed mice. (E) Heatmap representation of hepatic genes up- or downregulated by Nrg4 deficiency following NASH diet feeding (top). Enrichment of biological processes in these 2 clusters (bottom). (F) qPCR analysis of hepatic gene expression. Data represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001, 2-tailed unpaired Student’s t test. (G) Flow cytometry analysis of liver CD4⁺ T cells. Results are expressed as percentage (%) of IL-17⁺, TNF-α⁺, RORγt⁺, and FOXP3⁺ cells in total liver CD4⁺ T cells. Data represent mean ± SEM. *P < 0.05; **P < 0.01, 2-tailed unpaired Student’s t test.

Figure 3. Tg restoration of the Nrg4 endocrine axis protects mice from diet-induced NASH.

Control WT (n = 13, white) and Nrg4-Tg (n = 9, brown) male mice were fed a NASH diet for 20 weeks, starting at 3 months of age. (A) Plasma ALT, AST, and HMGB1 levels and liver TAG content. Data represent mean ± SEM. *P < 0.05, 2-tailed unpaired Student’s t test. (B) H&E, sirius red, F4/80 immunofluorescence, and TUNEL staining of liver sections. Scale bars: 100 μm. (C) Quantification of the sirius red, F4/80, and TUNEL staining images and liver hydroxyproline content. Data represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001, 2-tailed unpaired Student’s t test. (D) qPCR analysis of hepatic gene expression. Data represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001, 2-tailed unpaired Student’s t test. (E) Flow cytometry analysis of liver CD4⁺ T cells. Results are expressed as percentage (%) of IL-17⁺, TNF-α⁺, RORγt⁺, and FOXP3⁺ cells in total liver CD4⁺ T cells. Data represent mean ± SEM. *P < 0.05, 2-tailed unpaired Student’s t test. (F) Immunoblots of total liver lysates from NASH diet–fed mice.

Figure 4. Nrg4 signaling protects hepatocytes from stress-induced cell death.

(A) Immunoblots of total lysates from primary hepatocytes transduced with GFP or ErbB4 adenovirus and treated with Nrg4 for 20 minutes. (B) Immunoblots of total lysates from primary hepatocytes transduced with GFP or ErbB4 adenovirus treated with 150 μM PA for 2 hours followed by addition of 40 ng/ml TNF-α (PA/TNF-α) and 100 ng/ml Nrg4 for 20 hours. (C) LDH activity in culture media from hepatocytes transduced with Ad-ErbB4 and treated as indicated for 20 hours. Data represent mean ± SEM. **P < 0.01, 1-way ANOVA. (D) Flow cytometry analysis of hepatocytes (20 hours treatment) following annexin V and PI staining. Double-positive cells are considered dead, whereas annexin V–positive and PI-negative cells are apoptotic. (E) Quantitation of hepatocyte cell death based on annexin V/PI staining. Data represent mean ± SEM. ***P < 0.001, 1-way ANOVA. (F) LDH activity in culture media from Hepa 1 cells stably expressing ErbB4 and treated as indicated for 20 hours. Data represent mean ± SEM. **P < 0.01, 1-way ANOVA. (G) Flow cytometry analysis of cell death in treated Hepa 1 cells stably expressing ErbB4 (20 hours treatment). Data represent mean ± SEM. ***P < 0.001, 1-way ANOVA.

Figure 5. Stabilization of c-FLIP_L by Nrg4 attenuates stress-induced cell death in hepatoma cells.

The following experiments were performed in Hepa 1 cells stably expressing ErbB4. Cells were cultured in serum-free medium during treatment. (A) Immunoblots of total lysates from cells treated with PA/TNF-α in the absence or presence of 100 ng/ml Nrg4 for 6 hours. (B) Immunoblots of total lysates from cells treated with PA/TNF-α without or with 100 ng/ml Nrg4 and chased for different times in the presence of 2 μM CHX. (C) Immunoblots of total lysates from cells treated with PA/TNF-α without or with 100 ng/ml Nrg4 and chased for 6 hours in the presence of 10 μM MG132. (D) Immunoblots of total lysates from treated cells. Hepa 1 cells stably expressing ErbB4 were transduced with Ad-GFP or Ad–c-FLIP_L adenoviral vector. Transduced cells were treated with 100 μM PA for 2 hours followed by addition of 20 ng/ml TNF-α and 100 ng/ml Nrg4 for 20 hours. (E) LDH release by treated Hepa 1 cells (20 hours treatment). Data represent mean ± SEM. **P < 0.01, 1-way ANOVA. (F) Flow cytometry analysis (20 hours treatment). Data represent mean ± SEM. **P < 0.01; ***P < 0.001, 1-way ANOVA.

Figure 6. Nrg4 attenuates ubiquitination and proteasomal degradation of c-FLIP_L.

The following experiments were performed in Hepa 1 cells stably expressing ErbB4. Cells were cultured in serum-free medium during treatment. WCL, whole cell lysates. (A) Immunoblots of IP and whole cell lysates from Hepa 1 cells transfected with plasmids encoding HA-tagged c-FLIP_L and Flag-tagged ubiquitin (Flag-Ub) and treated with PA/TNF-α without or with 100 ng/ml Nrg4 for 4 hours. (B) Immunoblots of IP and whole cell lysates from transfected Hepa 1 cells treated in the absence or presence of AKT kinase inhibitor (20 μM) for 4 hours. (C) Immunoblots of total lysates from cells treated with PA/TNF-α without or with 100 ng/ml Nrg4 and in the absence or presence of AKT inhibitor (20 μM) for 6 hours. (D) LDH release by Hepa 1 cells as treated for 20 hours. Data represent mean ± SEM. ***P < 0.001, 1-way ANOVA. (E) Immunoblots of IP and WCL from transfected Hepa 1 cells treated in the absence or presence of JNK1/2 kinase inhibitor (20 μM) for 4 hours. (F) LDH release by Hepa 1 cells as treated for 20 hours. Data represent mean ± SEM. ***P < 0.001, 1-way ANOVA.

Figure 7. AAV-mediated restoration of hepatic c-FLIP_L alleviates NASH phenotype in Nrg4-deficient mice.

The following parameters were measured in WT and Nrg4-KO male mice fed NASH diet for a total of 20 weeks. AAV8-GFP and AAV8–c-FLIP_L vectors were administered 8 weeks following the initiation of NASH feeding. WT/GFP group, n = 8; WT/c-FLIP_L group, n = 7; Nrg4-KO/GFP group, n = 8; Nrg4 KO/c-FLIP_L group, n = 7. (A) Plasma ALT, AST, and HMGB1 levels. Data represent mean ± SEM. *P < 0.05, **P < 0.01, WT/GFP vs. Nrg4-KO/GFP; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, Nrg4-KO/GFP vs. Nrg4-KO/c-FLIP_L, 1-way ANOVA. (B) H&E, sirius red, F4/80 immunofluorescence, and TUNEL staining of liver sections. Scale bars: 100 μm. (C) Quantification of sirius red, F4/80, and TUNEL staining images and liver hydroxyproline content. Data represent mean ± SEM. *P < 0.05, **P < 0.01, WT/GFP vs. Nrg4 KO/GFP; ^##P < 0.01, ^###P < 0.001, Nrg4 KO/GFP vs. Nrg4 KO/c-FLIP_L, 1-way ANOVA.

Figure 8. AAV-mediated restoration of hepatic c-FLIP_L attenuates liver inflammation and cell death.

The following parameters were measured in mice as described in the Figure 7 legend. (A) qPCR analysis of hepatic gene expression. Data represent mean ± SEM. *P < 0.05, **P < 0.01, WT/GFP vs. Nrg4 KO/GFP; ^##P < 0.01, ^###P < 0.001, Nrg4 KO/GFP vs. Nrg4 KO/c-FLIP_L, 1-way ANOVA. (B) Immunoblots of total liver lysates. (C) Model depicting Nrg4-mediated endocrine signaling as a checkpoint for steatosis-to-NASH progression. Hepatic Nrg4 signaling counters c-FLIP_L downregulation in response to lipotoxic and inflammatory stress, thereby preserving hepatocyte health and blocking the transition from hepatic steatosis to NASH.