Hepatic neuregulin 4 signaling defines an endocrine checkpoint for steatosis-to-NASH progression (original) (raw)
Human and mouse NASH is linked to increased apoptosis and necroptosis in the liver. Hepatocyte death is at the center of the transition from relatively benign hepatic steatosis to NASH. The latter is characterized by persistent liver injury, chronic inflammation, and progressive liver fibrosis. Despite the prominent role of cell death in NASH pathogenesis, the physiological cues that preserve hepatocyte health and tissue homeostasis of the liver under metabolic stress conditions remain poorly understood. To address this, we first investigated the nature of hepatocyte death in human NASH patients and in a mouse model of diet-induced NASH. Compared with non-NASH individuals, the levels of cleaved caspase 3 (CASP3) and phosphorylated MLKL (p–MLKL-S358), molecular markers of apoptosis and necroptosis, respectively, were markedly elevated in liver biopsies from a cohort of NASH patients (33) (Figure 1A). The induction of cell death in NASH livers was associated with increased phosphorylation of JNK1/2 and reduced protein levels of c-FLIPL, an inhibitor of apoptotic and necroptotic cell death. Quantitative PCR (qPCR) gene-expression analysis indicated that mRNA expression of genes involved in inflammation and fibrosis was markedly increased in the liver from NASH patients (Figure 1B).
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.
We next evaluated a recently described high-fat high-fructose diet (NASH diet) in inducing key aspects of NASH pathology in mice (34). We fed a cohort of WT C57BL/6J mice standard chow or NASH diet for approximately 20 weeks. NASH diet–fed mice developed obesity and severe hepatic steatosis (Figure 1C and Supplemental Figure 1, A and B; supplemental material available online with this article; https://doi.org/10.1172/JCI96324DS1). Hepatic lipid droplet number and size were markedly increased following NASH diet feeding (Supplemental Figure 1, C and D). Compared with chow, plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were markedly higher in mice fed a NASH diet (Figure 1D). Plasma concentration of high-mobility group box 1 (HMGB1), a danger-associated molecular pattern released by dead cells, was also significantly higher in the NASH group than control (Figure 1D). Sirius red staining of liver sections indicated that NASH diet–fed mice developed pericellular liver fibrosis that was associated with induction of genes involved in inflammation and fibrosis, increased abundance of F4/80-positive macrophages, and the presence of TUNEL-positive cells (Figure 1, C and E, and Supplemental Figure 1, E and F). Immunoblotting analysis of liver lysates revealed that CASP3 and poly(ADP-ribose) polymerase (PARP) cleavage (apoptosis) and MLKL phosphorylation (necroptosis) were induced by NASH diet feeding (Figure 1F). Similarly to what occurred in human NASH livers, phosphorylation of JNK was increased, whereas c-FLIPL protein levels were reduced in the livers from NASH diet–fed mice. In contrast, protein levels of the short isoform c-FLIPR remained similar. These observations demonstrate that NASH diet feeding recapitulates key pathologic events of human NASH in mice, including liver injury, inflammation, and fibrosis in the context of obesity. Interestingly, mRNA expression of Nrg4 in epididymal white adipose tissue (eWAT) and brown adipose tissue (BAT) was significantly decreased in NASH mice (Figure 1G). These results are consistent with recent work that demonstrates an inverse relationship between plasma Nrg4 levels and NASH scores in human patients (27). As Nrg4 is a fat-derived endocrine factor that acts on the liver, these findings raise the possibility that reduced Nrg4 signaling may contribute to the disruption of tissue homeostasis during steatosis-to-NASH progression.
Nrg4 deficiency exacerbates diet-induced NASH in mice. We previously demonstrated that Nrg4 protects mice from high-fat diet–induced (HFD-induced) metabolic disorders, including insulin resistance and hepatic steatosis (25). However, HFD feeding does not cause significant liver injury and fibrosis and is therefore inadequate for interrogating the role of Nrg4 in NASH. We next performed studies in Nrg4 gain- and loss-of-function mouse models in the context of diet-induced NASH. WT and Nrg4-KO mice were fed standard chow or NASH diet for approximately 20 weeks. Hepatic steatosis and fibrosis, plasma ALT and AST, and mRNA expression of genes involved in liver fibrosis and inflammation were similar between 2 groups under chow-fed conditions (Supplemental Figure 2, A–D). Following NASH diet feeding, Nrg4-KO mice had slightly higher liver triacylglycerol (TAG) content than control (Figure 2A). While the number of lipid droplets in hepatocytes was comparable, lipid droplets were slightly larger in mice lacking Nrg4 (Supplemental Figure 2, E and F). The exacerbation of hepatic steatosis by Nrg4 deficiency was less marked compared with what was observed in mice fed HFD, which does not contain fructose and cholesterol. Plasma levels of ALT, AST, and HMGB1, the biomarkers of liver injury, were significantly elevated in Nrg4-KO mice following NASH feeding (Figure 2A). Nrg4-null mice exhibited more severe liver fibrosis, as revealed by sirius red staining and measurements of liver hydroxyproline content (Figure 2, B and C). Nrg4 deficiency also resulted in increased abundance of F4/80-positive macrophages and TUNEL-positive cells. Immunoblotting analysis indicated that CASP3 and PARP cleavage, MLKL phosphorylation, and JNK activation were strongly increased in Nrg4-KO mouse livers (Figure 2D). In contrast, c-FLIPL protein levels were markedly reduced by Nrg4 deficiency.
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.
Transcriptional profiling analysis identified 2 clusters of upregulated and downregulated genes in Nrg4-null mouse livers. Gene ontology analysis indicated that the upregulated genes are highly enriched for extracellular matrix, wound healing, and collagen, whereas the downregulated cluster was enriched for genes involved in lipid metabolism (Figure 2E). Consistently, mRNA expression of genes involved in liver fibrosis, including Col1a1, Acta2, Tgfb1, and Mmp13, and hepatic inflammation, including Tnfa, Il1b, Il12b, Nos2, Ccl2, Ccl5, and Adgre1 (F4/80), was significantly elevated in the livers from Nrg4-null mice (Figure 2F). These findings illustrate that Nrg4 deficiency worsened liver inflammation and fibrosis following NASH diet feeding. Th17 cells and IL-17 have recently been implicated in promoting steatohepatitis and liver fibrosis (35). Flow cytometry analysis indicated that the populations of Th17 cells and RORγt+CD4+ T cells were significantly increased in Nrg4-null mouse livers (Figure 2G). These results suggest that increased Th17 cell infiltration and IL-17 signaling may contribute to the exacerbation of NASH phenotypes in Nrg4-null mice. Together, these data demonstrate that Nrg4 signaling protects mice from diet-induced NASH and that its deficiency exacerbates key pathogenic events of NASH.
Tg restoration of the Nrg4 endocrine axis protects mice from NASH. Having established that Nrg4 deficiency is causally linked to NASH, we next explored whether Tg restoration of Nrg4 signaling is sufficient to ameliorate diet-induced NASH in mice. We fed WT and fat-specific Nrg4-Tg mice standard chow or NASH diet for approximately 20 weeks. Hepatic steatosis and fibrosis, plasma ALT and AST, and mRNA expression of genes involved in liver fibrosis and inflammation were similar between 2 groups following chow feeding (Supplemental Figure 3, A–D). Following NASH diet feeding, Nrg4-Tg mice had moderately lower liver fat content (Figure 3A), slightly smaller lipid droplets in hepatocytes (Supplemental Figure 3, E and F), and significantly lower plasma ALT, AST, and HMGB1 levels (Figure 3A), suggesting that Tg expression of Nrg4 may improve NASH diet–induced liver injury. In support of this, liver fibrosis and hydroxyproline content were significantly reduced in Tg mouse livers (Figure 3, B and C). Immunofluorescence staining revealed that the abundance of F4/80+ macrophage and TUNEL-positive cells was reduced in Tg mice, indicative of attenuated liver inflammation and hepatocyte death. Consistently, mRNA expression of genes involved in hepatic inflammation and fibrosis was significantly downregulated in Nrg4-Tg mouse livers, accompanied by reduced population of Th17 cells and RORγt+CD4+ T cells (Figure 3, D and E). Nrg4-Tg mouse livers exhibited reduced CASP3 and PARP cleavage and MLKL phosphorylation (Figure 3F). The levels of phosphorylated JNK were lower in Tg livers, whereas c-FLIPL protein levels were elevated in the livers of Nrg4-Tg mice. The strikingly opposite effects of Nrg4 deficiency and Tg expression on diet-induced NASH illustrate a crucial role of Nrg4 in serving as an endocrine checkpoint for steatosis-to-NASH progression. Given that Nrg4 manipulations only moderately affect the severity of hepatic steatosis, our results suggest that Nrg4 may exert cytoprotective functions on hepatocytes independently of its regulation of lipid metabolism.
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.
Nrg4 signaling protects hepatocytes from stress-induced cell death through c-FLIPL. Hepatocyte death is at the center of NASH initiation and progression, triggering chronic inflammation and fibrosis in the liver. The findings that liver injury was increased in Nrg4-null mice while attenuated in Nrg4-Tg mice prompted us to investigate whether Nrg4 directly modulates hepatocyte death. Previous work has demonstrated that liver expresses all ErbB receptors except ErbB2 (36). However, ErbB4 expression in primary hepatocyte culture is relatively low. We reconstituted Nrg4/ErbB4 signaling by transducing primary hepatocytes with a recombinant adenoviral vector expressing ErbB4, as previously described (25). As expected, recombinant Nrg4 dose-dependently stimulated ErbB4 receptor tyrosine phosphorylation and AKT phosphorylation in transduced primary hepatocytes (Figure 4A). NASH-associated hepatocyte death results from an array of stress signals, most notably lipotoxicity and proinflammatory cytokines. Previous studies have demonstrated that treatments of primary hepatocytes with a combination of palmitic acid (PA) and TNF-α (PA/TNF-α) robustly triggered cell death that mimics NASH-induced liver injury (21, 37). To investigate whether Nrg4 plays a direct role in the regulation of hepatocyte death, we treated primary hepatocytes transduced with GFP or ErbB4 adenovirus with PA/TNF-α. As expected, PA/TNF-α treatment increased CASP3 and PARP cleavage and MLKL phosphorylation in hepatocytes transduced with Ad-GFP, indicative of induction of apoptotic and necroptotic cell death in response to PA and TNF-α (Figure 4B). Nrg4 treatment strongly attenuated cell death markers in primary hepatocytes expressing ErbB4. We measured the release of lactate dehydrogenase (LDH) into culture media as an indicator of hepatocyte death (38). LDH release by primary hepatocytes was markedly increased in response to PA/TNF-α treatment (Figure 4C). Activation of Nrg4 signaling strongly attenuated LDH release induced by PA/TNF-α. Further, flow cytometry analysis using annexin V and propidium iodide (PI) costaining indicated that PA/TNF-α treatment increased the frequency of apoptotic (annexin V positive and PI negative) and dead (double positive) cells, which was significantly alleviated by Nrg4 (Figure 4, D and E). Similar protective effects of Nrg4 were also observed in transduced Hepa 1 hepatoma cells stably expressing ErbB4 (Figure 4, F and G, and Supplemental Figure 4, A–C).
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.
c-FLIPL is a cell death inhibitory protein that has been shown to suppress both apoptosis and necroptosis and protect against liver injury in mice (39–41). As shown above, c-FLIPL protein level was reduced in Nrg4-null mouse livers, but elevated by Nrg4-Tg expression in adipose tissue. These observations suggest that c-FLIPL may be a downstream effector of Nrg4 that links Nrg4/ErbB4 signaling to hepatocyte death. Interestingly, c-FLIP mRNA expression in Nrg4-null and -Tg mouse livers remained largely unchanged (Figure 2F and Figure 3D), suggesting that Nrg4 may regulate c-FLIPL protein levels through posttranslational mechanisms. In support of this, we found that endogenous c-FLIPL protein levels were reduced in hepatoma cells in response to PA/TNF-α treatment without affecting c-Flip mRNA levels (Figure 5A and Supplemental Figure 5A). Nrg4 treatment stimulated AKT phosphorylation, attenuated JNK phosphorylation, and nearly completely blocked the decrease of c-FLIPL protein expression by PA/TNF-α treatment. Cycloheximide (CHX) chase studies indicated that Nrg4 increased the half-life of c-FLIPL in hepatoma cells (Figure 5B). Accordingly, the proteasome inhibitor MG132 blocked PA/TNF-α–induced decrease of c-FLIPL (Figure 5C), consistent with a posttranslational mechanism of regulation.
Stabilization of c-FLIPL 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-FLIPL 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.
We next performed studies in Hepa 1 cells stably expressing ErbB4 using a retroviral vector to dissect the role of c-FLIPL in mediating the cytoprotective activity of Nrg4. Interestingly, c-FLIPL protein level was markedly reduced, whereas CASP3 cleavage and MLKL phosphorylation were stimulated, in response to PA/TNF-α treatment (Figure 5D). The effects of PA/TNF-α on c-FLIPL protein level and cell death markers were partially reversed by Nrg4 treatment. Further, adenovirus-mediated expression of c-FLIPL greatly diminished PA/TNF-α–induced apoptotic and necroptotic cell death. These results were confirmed by LDH release and flow cytometry analyses (Figure 5, E and F, and Supplemental Figure 4D). We did not observe further improvement of cell viability by Nrg4 in c-FLIPL–overexpressing hepatoma cells, suggesting that the protective effects of Nrg4 are largely mediated by its induction of c-FLIPL protein expression.
Nrg4 attenuates ubiquitination and proteasomal degradation of c-FLIPL. We performed ubiquitination assay to determine whether PA/TNF-α and Nrg4 play an antagonistic role in c-FLIPL ubiquitination. As shown in Figure 6A, PA/TNF-α treatment induced robust polyubiquitination of c-FLIPL that was largely reversed by Nrg4 treatment. We next determined whether AKT activation by Nrg4 is required for its regulation of c-FLIPL protein stability. We performed PA/TNF-α and Nrg4 treatments without or with AKTi, a specific AKT inhibitor. The ability of Nrg4 to attenuate c-FLIPL ubiquitination in response to PA/TNF-α treatment was greatly diminished in the presence of AKTi (Figure 6B). As a result, Nrg4 failed to stabilize c-FLIPL protein and suppress PA/TNF-α–induced cell death, as shown by CASP3 cleavage, MLKL phosphorylation, and LDH release (Figure 6, B–D). On the contrary, inhibition of JNK kinase activity blocked the induction of c-FLIPL ubiquitination by PA/TNF-α treatment, leading to improved cell viability (Figure 6, E and F). Nrg4 treatment did not further decrease c-FLIPL ubiquitination. As such, Nrg4 exerts opposing effects on the AKT and JNK signaling pathways to regulate c-FLIPL ubiquitination and proteasomal degradation in response to PA/TNF-α. Further, the reciprocal regulation of c-FLIPL protein levels in Nrg4-KO and -Tg mouse livers likely reflects cell-autonomous action of Nrg4 in hepatocytes.
Nrg4 attenuates ubiquitination and proteasomal degradation of c-FLIPL. 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-FLIPL 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.
Previous work has identified Itchy E3 ubiquitin protein ligase (Itch) as a JNK-regulated E3 ubiquitin ligase for c-FLIPL (42). To investigate whether Itch is required for ubiquitination and proteasomal degradation of c-FLIPL in response to PA/TNF-α, we knocked down Itch in Hepa 1 cells using 2 independent siRNA pools against Itch. Surprisingly, siRNA knockdown of Itch did not appear to affect c-FLIPL ubiquitination and degradation and PA/TNF-α–induced cell death (Supplemental Figure 5, B and C). These results suggest that it is unlikely that Itch is the E3 ubiquitin ligase responsible for PA/TNF-α–induced c-FLIPL ubiquitination.
Restoration of hepatic c-FLIPL expression blocks steatosis-to-NASH progression due to Nrg4 deficiency. The above studies illustrate the nature of molecular signaling that links Nrg4 signaling to regulation of c-FLIPL protein stability and hepatocyte cell death. A key prediction of this model is that restoration of hepatic c-FLIPL protein expression will improve diet-induced NASH in mice due to Nrg4 deficiency. To test this, we generated a c-FLIPL expression vector using adeno-associated virus (AAV) type 8 under the control of the liver-specific thyroid hormone-binding globulin (Tbg) promoter. Tail vein injection of AAV–c-FLIPL elevated c-FLIPL expression specifically in the liver, but not other tissues, such as adipose tissue, spleen, and lung (Supplemental Figure 6A). We fed WT and Nrg4-null mice NASH diet for 8 weeks and transduced these mice with control AAV-GFP or AAV–c-FLIPL virus. Four treatment groups were obtained: WT mice injected with AAV-GFP (WT/GFP) or AAV–c-FLIPL (WT/c-FLIPL) and Nrg4-KO mice injected with AAV-GFP (Nrg4 KO/GFP) or AAV–c-FLIPL (Nrg4 KO/c-FLIPL). We examined NASH features in transduced mice following an additional 12 weeks of NASH diet feeding. Similar to what occurred in the studies shown in Figure 2, Nrg4 deficiency accelerated diet-induced NASH in mice. Compared with WT/GFP, Nrg4-KO/GFP mice had higher plasma ALT, AST, and HMGB1 levels, developed more severe liver fibrosis, and exhibited increased presence of apoptotic cells (Figure 7, A–C). While AAV-mediated overexpression of c-FLIPL moderately improved NASH parameters in WT mice, it nearly completely eliminated worsening of NASH in Nrg4-null mice. Hepatic gene expression analyses indicated that the expression of genes involved in inflammation and liver fibrosis in Nrg4-null mice was greatly attenuated by AAV–c-FLIPL, reaching levels similar to those in WT mice (Figure 8A). Accordingly, CASP3 and PARP cleavage and MLKL phosphorylation in Nrg4-KO mice also returned to control levels following transduction with AAV–c-FLIPL (Figure 8B). Liver fat content and hepatic lipid droplet number and size, however, were not significantly affected by AAV-mediated overexpression of c-FLIPL (Supplemental Figure 6, B–D). These results support a critically important functional role of c-FLIPL regulation by Nrg4 in mediating its effects on NASH progression.
AAV-mediated restoration of hepatic c-FLIPL 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-FLIPL vectors were administered 8 weeks following the initiation of NASH feeding. WT/GFP group, n = 8; WT/c-FLIPL group, n = 7; Nrg4-KO/GFP group, n = 8; Nrg4 KO/c-FLIPL 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-FLIPL, 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-FLIPL, 1-way ANOVA.
AAV-mediated restoration of hepatic c-FLIPL 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-FLIPL, 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-FLIPL downregulation in response to lipotoxic and inflammatory stress, thereby preserving hepatocyte health and blocking the transition from hepatic steatosis to NASH.