Abstract

Background & aims

   Extracellular nicotinamide phosphoribosyltransferase (eNAMPT) has long
   been recognized as an adipokine. However, the exact role of eNAMPT in
   alcoholic liver disease (ALD) and its relevance to brown adipose tissue
   (BAT) remain largely unknown. This study aimed to evaluate the impact
   of eNAMPT on liver function and the underlying mechanisms involved in
   BAT-Liver communication.

Methods

   Serum eNAMPT levels were detected in the serum of both ALD patients and
   mice. Chronic and binge ethanol feeding was used to induce alcoholic
   liver injury in mice. An eNAMPT antibody, a coculture model of brown
   adipocytes and hepatocytes, and BAT-specific Nampt knockdown mice were
   used to investigate the role of eNAMPT in ALD.

Results

   Serum eNAMPT levels are elevated in ALD patients and are significantly
   positively correlated with the liver injury index. In ALD mice,
   neutralizing eNAMPT reduced the elevated levels of circulating eNAMPT
   induced by ethanol and attenuated liver injury. In vitro experiments
   revealed that eNAMPT induced hepatocyte ferroptosis through the
   TLR4-dependent mitochondrial ROS-induced ferritinophagy pathway.
   Furthermore, ethanol stimulated eNAMPT secretion from brown adipocytes
   but not from other adipocytes. In the coculture model, ethanol-induced
   release of eNAMPT from brown adipocytes promoted hepatocyte
   ferroptosis. In BAT-specific Nampt-knockdown mice, ethanol-induced
   eNAMPT secretion was significantly reduced, and alcoholic liver injury
   were attenuated. These effects can be reversed by intraperitoneal
   injection of eNAMPT.

Conclusion

   Inhibition of ethanol-induced eNAMPT secretion from BAT attenuates
   liver injury and ferroptosis. Our study reveals a previously
   uncharacterized critical role of eNAMPT-mediated BAT-Liver
   communication in ALD and highlights its potential as a therapeutic
   target.

   Keywords: eNAMPT, Mitochondrial dysfunction, Ferroptosis, Brown adipose
   tissue, Alcoholic liver injury

Graphical abstract

   [59]Image 1
   [60]Open in a new tab

1. Introduction

   Alcoholic liver disease (ALD) is a common chronic liver disease caused
   by chronic and excessive alcohol consumption [[61]1]. The prevalence of
   ALD has increased in recent years due to increased alcohol consumption
   [[62]2,[63]3]. The half-life of alcohol in the human body is 4–5 h, so
   alcohol is completely eliminated within a few hours to a few days,
   depending on the activity of acetaldehyde dehydrogenase and gut
   microbiota [[64]4,[65]5]. However, alcohol-induced damages to the liver
   and other organs have long-lasting or even permanent effects,
   suggesting that the indirect effects of alcohol have a major impact on
   the development of ALD. Alcohol consumption has been shown to cause
   dysfunction in adipose tissue, affecting the secretion of adipokines,
   proinflammatory mediators, and free fatty acids [[66]6]. Studies have
   reported that alcohol abstinence reverses adipose tissue dysfunction
   [[67]7], and the normalization of adipose tissue function through
   rosiglitazone treatment attenuates alcoholic liver injury
   [[68]8,[69]9]. These studies suggested that the intricate communication
   between adipose tissues and the liver may modulate alcoholic liver
   injury, providing new insights for ALD therapy.

   Nicotinamide phosphoribosyltransferase (NAMPT), also known as pre-B
   colony-enhancing factor or visfatin, exists in two forms: intracellular
   NAMPT (iNAMPT) and extracellular NAMPT (eNAMPT). iNAMPT is highly
   expressed in brown adipose tissue (BAT) [[70]10]. eNAMPT is secreted by
   adipocytes as an adipokine [[71]11,[72]12]. iNAMPT is the rate-limiting
   enzyme for nicotinamide adenine dinucleotide (NAD) synthesis. This
   enzyme is involved in cell metabolism and physiological activities and
   has been well recognized by numerous studies [[73]13,[74]14]. However,
   the pathophysiological roles of eNAMPT are diverse and controversial.
   eNAMPT functions as a cytokine that induces an oxidative stress
   response and apoptosis, as a proinflammatory mediator that induces
   inflammation, and as an NAD synthesis enzyme that extends the lifespan
   in mice [[75]15,[76]16]. The receptor of eNAMPT has not been fully
   identified, and several studies have shown that eNAMPT can directly
   bind to Toll-like receptor 4 (TLR4) [[77]17,[78]18]. Indeed, the
   important role of eNAMPT has been highlighted in various diseases, such
   as obesity, atherosclerosis, diabetes, aging, and cancer [[79]15].
   eNAMPT can induce reactive oxygen species (ROS) production in several
   cells [[80][19], [81][20], [82][21]]. Hepatic ROS production,
   mitochondrial dysfunction, and lipid peroxidation have been implicated
   in the pathogenesis of ALD and may lead to the death of hepatocytes,
   liver injury, and inflammation [[83]22,[84]23]. However, the
   pathophysiological effects of eNAMPT on the progression of ALD,
   particularly the role of BAT-derived eNAMPT in the liver, have not been
   elucidated. It is necessary to improve the understanding of
   eNAMPT-related pathways and mechanisms in ALD.

   Ferroptosis is a form of iron-dependent programmed cell death
   characterized by excessive oxidative stress and lipid peroxidation
   [[85]24]. Ferritin heavy chain 1 (FTH1) is one of the major iron
   storage proteins in the body and combines with the ferritin light chain
   to form the ferritin complex, which maintains cellular free iron
   homeostasis [[86]25]. Recently, studies have shown that ferritinophagy
   can promote ferroptosis by degrading ferritin, leading to an increase
   in iron levels, which triggers the Fenton reaction to generate
   excessive ROS, leading to lipid peroxidation and ultimately cell death
   [[87][25], [88][26], [89][27]]. In addition, mitochondria are known to
   be major organelles involved in ROS production [[90]28]. Morphological
   damage to mitochondria can be observed in cells undergoing ferroptosis
   [[91]29]. However, the exact role of mitochondria in the regulation of
   ferroptosis has not been determined. Ferroptosis is also characterized
   by the depletion of glutathione (GSH), which is a cofactor for
   glutathione peroxidase 4 (GPX4) and plays a critical role in the
   suppression of lipid peroxidation and ferroptosis [[92]29,[93]30].
   Previous research has suggested that ferroptosis plays an emerging role
   in the development of ALD [[94]31,[95]32]. However, the specific
   mechanism of ferroptosis in ALD remains to be explored.

   In this study, we hypothesized that eNAMPT secreted from BAT plays a
   critical role in regulating the progression of ALD. To test this
   hypothesis, we applied the loss and gain of function of eNAMPT to
   confirm its function. We neutralized eNAMPT with an eNAMPT antibody in
   mice, generated BAT-specific Nampt-knockdown mice, and further supplied
   eNAMPT directly. Interestingly, we showed that alcohol-induced eNAMPT
   secretion from brown adipocytes induces liver ferroptosis through
   TLR4-dependent ferritinophagy via the BAT-Liver axis. Our study
   revealed a previously uncharacterized critical role of eNAMPT-mediated
   BAT-Liver communication in ALD and revealed its potential as a
   therapeutic target.

2. Results

2.1. The serum eNAMPT concentration is elevated in both ALD patients and
ethanol-fed mice

   To determine whether circulating eNAMPT is potentially involved in ALD,
   we examined the serum eNAMPT concentration in both ALD patients and
   ethanol-fed mice. The results showed that eNAMPT was significantly
   increased in ALD patients compared to healthy individuals ([96]Fig.
   1A). In addition, the increase in the serum eNAMPT concentration was
   positively correlated with the serum alanine aminotransferase (ALT)
   (r = 0.7314, p = 0.0008) and aspartate aminotransferase (AST)
   (r = 0.6646, p = 0.0036) levels ([97]Fig. 1A). Similarly, compared with
   control mice, ethanol-fed mice also presented significantly increased
   serum eNAMPT levels, which were positively correlated with ALT
   (r = 0.7146, p = 0.0013) and AST (r = 0.7318, p = 0.0008) levels
   ([98]Fig. S1A). These results suggest that elevated levels of eNAMPT
   may contribute to the development of alcoholic liver injury.

Fig. 1.

   [99]Fig. 1
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   Neutralizing eNAMPT protects against alcoholic liver injury and
   regulates mitochondrial oxidative phosphorylation

   (A) Serum was collected from healthy individuals and ALD patients, and
   the serum eNAMPT levels in healthy controls and ALD patients (n = 17)
   were measured via ELISA and compared between the serum eNAMPT and ALT
   or AST. (B) Schematic of the experimental design for mice treated with
   chronic-plus-binge ethanol. Treatment with the NAMPT neutralizing
   antibody was administered every three days. (C) The serum eNAMPT, AST
   and ALT levels were measured. (D) F4/80 expression in mouse liver was
   determined by immunohistochemistry. (E–G) mRNA-seq sequencing of mouse
   livers from the EtOH and EtOH + Anti-NAMPT groups. (E) Gene set
   enrichment analysis, (F) KEGG pathway enrichment analysis and (G)
   expression heatmap. The data are expressed as the means ± SEMs
   (n = 6–9/group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001,
   statistical analysis by One-way ANOVA or Pearson correlation analysis.

2.2. Neutralizing eNAMPT alleviates alcoholic liver injury and regulates
hepatic mitochondrial oxidative phosphorylation in mice

   To investigate the role of eNAMPT in alcoholic liver injury, we used a
   polyclonal NAMPT antibody (Anti-NAMPT) to neutralize circulating eNAMPT
   in a mouse model of chronic and binge ethanol feeding. Anti-NAMPT was
   administered by intraperitoneal injection every three days ([101]Fig.
   1B). Body weight and food consumption were not significantly different
   between the control and treatment groups ([102]Figs. S1C–D). Our
   results showed that Anti-NAMPT treatment significantly decreased the
   serum level of eNAMPT and reduced the serum AST and ALT in ethanol-fed
   mice ([103]Fig. 1C). Immunohistochemical analysis of ethanol-fed mice
   revealed that Anti-NAMPT treatment decreased the expression of hepatic
   F4/80 (a marker of macrophages) ([104]Fig. 1D). Histological H&E
   staining of the liver revealed that Anti-NAMPT treatment didn't change
   the ethanol-induced hepatic lipid accumulation ([105]Figs. S1E–F).
   These results suggest that neutralizing eNAMPT may attenuate alcoholic
   liver injury without affecting steatosis. To further understand the
   mechanism of action of eNAMPT in the liver, we performed RNA sequencing
   analysis. RNA-seq analysis between EtOH and EtOH + Anti-NAMPT groups
   revealed 278 upregulated genes and 363 downregulated genes
   (differentially expressed genes, DEGs) after Anti-NAMPT treatment
   ([106]Fig. S2A). Both gene set enrichment analysis (GSEA) and Kyoto
   Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that
   the DEGs were highly enriched in gene sets associated with the
   mitochondrial oxidative phosphorylation and ferroptosis pathways
   ([107]Fig. 1E–F). In addition, Gene Ontology analysis of cellular
   components revealed that the DEGs were enriched in mitochondria
   ([108]Fig. S2B). Notably, the gene expression heatmap showed that EtOH
   treatment down-regulated the expression of mitochondrial electron
   transport chain subunit genes compared with the control group in mouse
   liver ([109]Fig. 1G). However, Anti-NAMPT treatment upregulated the
   expression of these mitochondrial genes ([110]Fig. 1G). Furthermore,
   interaction analysis of the DEGs suggested that Cox6a1, Ndufa2, and
   Uqcr11 were strongly correlated with the effect of Anti-NAMPT treatment
   ([111]Fig. S2C). These results suggest that neutralizing eNAMPT can
   alleviate alcoholic liver injury and regulate hepatic mitochondrial
   oxidative phosphorylation in mice.

2.3. Neutralizing eNAMPT suppresses hepatic mitochondrial dysfunction and
ferroptosis in mice

   To verify the effects of eNAMPT on alcoholic liver injury, we evaluated
   mitochondrial function and ferroptosis in the mouse liver. Notably,
   immunohistochemical analysis revealed that Anti-NAMPT treatment
   increased the expression of TOM20 (a mitochondrial membrane protein) in
   ethanol-fed mice ([112]Fig. 2A). Treatment with Anti-NAMPT
   significantly increased the hepatic mRNA expression of the Cox6a1,
   Ndufa2, Atp5j, and Uqcr11 genes in ethanol-fed mice ([113]Fig. 2B).
   Ethanol metabolism disrupts the NAD pool and affects the oxidative
   reaction of the mitochondrial electron transport chain for ATP
   synthesis [[114]33]. Our results showed significantly decreased hepatic
   NAD levels and the NAD^+/NADH ratio, accompanied by decreased ATP
   levels and ATP/ADP ratio and increased AMP/ATP ratio in ethanol-fed
   mice compared with control mice ([115]Fig. 2C). However, Anti-NAMPT
   treatment restored NAD and ATP levels and improved energy homeostasis,
   indicating that neutralization of eNAMPT can ameliorate ethanol-induced
   mitochondrial dysfunction ([116]Fig. 2C). In addition, Anti-NAMPT
   treatment significantly decreased the levels of MDA and the expression
   of 4-HNE in the livers of ethanol-fed mice ([117]Fig. 2D–E). Next, we
   detected the expression of specific ferroptosis-related proteins in the
   liver, including GPX4, FTH1, ACSL4, and COX2 [[118]34,[119]35]. Ethanol
   supplementation decreased the protein expression of GPX4 and FTH1;
   increased the protein expression of ACSL4 and COX2; decreased the
   hepatic GSH/GSSG ratio; and increased hepatic iron levels ([120]Fig.
   2F–H), indicating that liver ferroptosis was activated in ethanol-fed
   mice. However, Anti-NAMPT treatment significantly reversed the changes
   in the expression of these specific ferroptosis-related proteins
   ([121]Fig. 2F) and attenuated hepatic GSH depletion and iron
   accumulation in the livers of ethanol-fed mice ([122]Fig. 2G–H). In
   addition, Anti-NAMPT treatment significantly decreased the mRNA
   expression of Ncoa4, Atg5, and Acsl4 and increased the mRNA expression
   of Nrf 2, Fth1, and Gpx4 in the livers of ethanol-fed mice ([123]Fig.
   2I). These results suggest that neutralizing eNAMPT can suppress
   hepatic mitochondrial dysfunction and ferroptosis in alcoholic liver
   injury.

Fig. 2.

   [124]Fig. 2
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   Neutralizing eNAMPT protects against liver mitochondrial dysfunction
   and ferroptosis in ethanol-fed mice

   Mice were treated with chronic-plus-binge ethanol feeding and
   NAMPT-neutralizing antibody for 10 days. (A) Expression of TOM20 in the
   mouse liver was determined by immunohistochemistry. (B) Hepatic mRNA
   levels of complex subunits of mitochondrial respiratory chain genes
   were measured by qRT‒PCR; (C) Liver NAD and ATP levels, ATP/ADP ratio,
   AMP/ATP ratio, and the NAD+/NADH ratio were measured by UPLC‒QTOF. (D)
   Expression of 4-HNE in the mouse liver was determined by
   immunohistochemistry. (E) The levels of MDA were measured in the mouse
   liver. (F) The protein expression levels of GPX4, FTH1, ACSL4, and COX2
   in the mouse liver were measured via Western blotting, and the results
   are expressed as fold changes relative to the control. (G) The GSH/GSSG
   ratio was measured in the mouse liver. (H) The Fe^2+ concentration was
   measured in the mouse liver. (I) Hepatic mRNA levels of ferroptosis
   genes were measured via qRT‒PCR. The data are expressed as the
   means ± SEMs (n = 6–9/group); ^&p < 0.05 compared with the EtOH group;
   ^#p < 0.05 compared with the control group according to One-way ANOVA.
   *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

2.4. eNAMPT induces mitochondrial oxidative stress and ferroptosis in
hepatocytes in vitro

   To further investigate the effect of eNAMPT on hepatocytes, we
   incubated AML12 cells or HepG2 cells with recombinant eNAMPT (5 ng/mL
   and 10 ng/mL) for 24 h. Our results showed that the oxygen consumption
   rate (OCR) was significantly decreased by eNAMPT treatment in a
   dose-dependent manner in AML12 cells ([126]Fig. 3A) and HepG2 cells
   ([127]Fig. S3A). In addition, eNAMPT treatment also significantly
   increased the ROS levels in a dose-dependent manner, the recombinant
   eNAMPT we purified has the same effect on ROS production in hepatocytes
   compared with commercial recombinant eNAMPT ([128]Fig. S4A). Therefore,
   10 ng/mL eNAMPT was selected for the following study. Electron
   microscopy analysis revealed structural changes in mitochondria after
   eNAMPT treatment, including a broken and wrinkled outer membrane,
   reduced or absent mitochondrial cristae, and shrunken mitochondria
   ([129]Fig. 3B). eNAMPT treatment increased mitochondrial ROS (MtROS)
   levels, as shown by Mito-SOX staining ([130]Fig. 3C). These results
   indicate that eNAMPT induces mitochondrial dysfunction and oxidative
   stress. In addition, eNAMPT treatment elevated the levels of MDA and
   was accompanied by an increase in the level of Liperfluo staining,
   indicating increased lipid peroxidation in AML12 cells ([131]Fig.
   3D–E). Similarly, after eNAMPT exposure, the GSH/GSSG ratio and the
   GPX4 expression decreased, while the COX2 expression increased
   ([132]Fig. 3F–G). Importantly, treatment with eNAMPT significantly
   decreased the viability of AML12 cells ([133]Fig. 3H and [134]Fig.
   S4B). To confirm whether eNAMPT can induce ferroptosis, we incubated
   AML12 cells with Fer-1, a specific inhibitor of ferroptosis. As
   expected, Fer-1 treatment reversed the eNAMPT-induced decrease in GPX4
   and the depletion of GSH, inhibited the increase in COX2 and MDA
   levels, and eventually inhibited cell death ([135]Fig. 3I–K). These
   results indicate that eNAMPT induces mitochondrial oxidative stress and
   ferroptosis in hepatocytes.

Fig. 3.

   [136]Fig. 3
   [137]Open in a new tab

   eNAMPT induces mitochondrial oxidative stress and ferroptosis in
   hepatocytes

   (A) AML12 cells were incubated with recombinant NAMPT protein at
   5 ng/mL and 10 ng/mL for 24 h, after which the oxygen consumption rate
   (OCR) was measured, statistical analysis by One-way ANOVA. (B–H)
   AML12 cells were incubated with recombinant eNAMPT protein at 10 ng/mL,
   statistical analysis by Student's t-test. (B) Representative
   transmission electron microscopy images. (C–D) Representative
   immunofluorescence images of MitoSOX staining and Liperfluo staining.
   (E) The levels of MDA were measured in AML12 cells. (F) The ratio of
   GSH/GSSG were measured in AML12 cells. (G) The expression of GPX4 and
   COX2 was measured by Western blot. The results are expressed as fold
   changes relative to the control. (H) AML12 cell viability was measured
   via a CCK8 kit. (I–K) AML12 cells were incubated with recombinant NAMPT
   protein at 10 ng/mL and treated with or without Fer-1 (100 nM, a
   ferroptosis-specific inhibitor) for 24 h, statistical analysis by
   Two-way ANOVA. (I) The expression of the GPX4 and COX2 proteins were
   measured via Western blotting, (J) the GSH/GSSG ratio and MDA levels
   were measured in AML12 cells; the results are expressed as fold changes
   relative to the control, (K) the cell viability was measured via a CCK8
   kit. The data are expressed as the means ± SEMs of three independent
   experiments. ^&p < 0.05 compared with the eNAMPT treatment group;
   ^#p < 0.05 compared with the DMSO group; *p < 0.05, **p < 0.01,
   ***p < 0.001, ****p < 0.0001.

2.5. eNAMPT induces hepatocyte ferroptosis through mitochondrial ROS-induced
ferritinophagy

   Iron homeostasis is the most important factor influencing the
   development of ferroptosis [[138]36]. Treatment with eNAMPT
   significantly increased iron levels and the expression of LC3-Ⅱ and
   decreased the expression of p62 and ferritin in AML12 cells ([139]Fig.
   4A–B) and HepG2 cells ([140]Fig. S3E), suggesting that eNAMPT might
   induce ferritinophagy. To confirm the effect of eNAMPT on
   ferritinophagy, we treated AML12 cells with the autophagy inhibitor
   hydroxychloroquine (HCQ). The results showed that HCQ inhibited the
   increase in iron levels and the decrease in ferritin expression induced
   by eNAMPT ([141]Fig. 4C–D). HCQ treatment enhanced the increase in
   LC3-Ⅱ expression but inhibited the decrease in p62, and the expression
   of ferritin was restored in AML12 cells ([142]Fig. 4D). These results
   indicate that eNAMPT-induced iron accumulation depends on the
   activation of ferritinophagy. HCQ also reversed eNAMPT-induced cell
   death and excessive production of total intracellular ROS in
   AML12 cells ([143]Fig. 4E). These results demonstrated that eNAMPT can
   disturb iron homeostasis in hepatocytes by regulating ferritinophagy.
   To explore the correlation between iron homeostasis and mitochondrial
   oxidative stress and the effect of eNAMPT, we treated AML12 and HepG2
   cells with the MtROS inhibitor Mito-TEMPO ([144]Figs. S3C–E). The
   results showed that Mito-TEMPO significantly inhibited the
   eNAMPT-induced increase in MtROS production ([145]Fig. 4F).
   Interestingly, Mito-TEMPO inhibited the eNAMPT-induced increase in the
   expression of LC3-Ⅱ and the decrease in the expression of p62 and
   ferritin, ultimately inhibiting the increase in iron levels in
   AML12 cells ([146]Fig. 4G–H). As expected, total intracellular ROS were
   inhibited by Mito-TEMPO compared to treatment with eNAMPT alone
   ([147]Fig. 4H). eNAMPT-induced lipid peroxidation was also inhibited by
   Mito-TEMPO, as shown by the MDA levels and Liperfluo staining of
   AML12 cells ([148]Fig. 4I–J). Furthermore, Mito-TEMPO treatment
   reversed the GSH depletion, increased the expression of GPX4, decreased
   the expression of COX2 ([149]Fig. 4K–L), and ultimately inhibited the
   death of AML12 cells induced by eNAMPT ([150]Fig. 4L). These results
   indicate that eNAMPT induces hepatocyte ferroptosis by promoting
   MtROS-mediated ferritinophagy.

Fig. 4.

   [151]Fig. 4
   [152]Open in a new tab

   eNAMPT induces hepatocyte ferroptosis through mitochondrial ROS-induced
   ferritinophagy

   (A-B) AML12 cells were incubated with recombinant eNAMPT protein at
   10 ng/mL, statistical analysis by Student's t-test. (A) The protein
   expression levels of ferritin, p62 and LC3 were measured via Western
   blotting. The results are expressed as fold changes relative to the
   control. (B) Cellular Fe^2+ levels were measured in AML12 cells. (C–E)
   AML12 cells were incubated with recombinant eNAMPT protein at 10 ng/mL
   and treated with or without hydroxychloroquine (HCQ, 10 μM, an
   autophagy inhibitor) for 24 h, statistical analysis by Two-way ANOVA.
   (C) Cellular Fe^2+ levels were measured in AML12 cells. (D) The protein
   expression levels of ferritin, p62 and LC3 were measured via Western
   blotting. The results are expressed as fold changes relative to the
   control. (E) Intracellular ROS levels were detected with the
   fluorescent probe DCFH-DA, and the fluorescence intensity was
   subsequently measured. Cell viability was analyzed by a CCK8 kit. (F–L)
   AML12 cells were incubated with recombinant eNAMPT protein at 10 ng/mL
   and treated with or without Mito-TEMPO (100 nM, a mitochondrial ROS
   inhibitor) for 24 h, statistical analysis by Two-way ANOVA. (F)
   Representative immunofluorescence images of MitoSOX staining. (G) The
   expression of FTH1, p62, and LC3 was measured by Western blotting. The
   results are expressed as fold changes relative to the control. (H)
   Cellular Fe^2+ levels were measured in AML12 cells. Intracellular ROS
   levels were detected by the fluorescent probe DCFH-DA. (I)
   Representative immunofluorescence images of Liperfluo staining. (I)
   Cellular MDA levels were measured in AML12 cells. (K) The expression of
   GPX4 and COX2 was measured via Western blotting. The results are
   expressed as fold changes relative to the control. (L) The GSH/GSSG
   ratio was measured in AML12 cells. Cell viability was measured in
   AML12 cells by a CCK8 kit. The data are expressed as the means ± SEMs
   from three independent experiments. *p < 0.05 compared with the eNAMPT
   treatment group; ^#p < 0.05 compared with the CON group; *p < 0.05,
   **p < 0.01, ***p < 0.001, ****p < 0.0001.

2.6. Hepatocyte ferroptosis induced by eNAMPT is mediated by TLR4

   Previous studies suggest that eNAMPT may bind to TLR4 and regulate
   cellular functions [[153]17,[154]37,[155]38]. We hypothesized that
   eNAMPT-induced MtROS production might be mediated by TLR4. To test this
   hypothesis, we treated AML12 and HepG2 cells with a TLR4 inhibitor
   (Resatorvid) ([156]Figs. S3B–E). As expected, Resatorvid treatment
   effectively prevented the decrease in the OCR induced by eNAMPT
   ([157]Fig. 5A) and significantly reduced the eNAMPT-induced excessive
   production of MtROS ([158]Fig. 5B), indicating that eNAMPT-induced
   mitochondrial dysfunction is dependent on TLR4. Resatorvid inhibited
   the eNAMPT-induced increase in the expression of LC3-Ⅱ and the decrease
   in the expression of p62 and ferritin and inhibited the eNAMPT-induced
   increase in iron levels in AML12 cells ([159]Fig. 5C–D). In addition,
   Resatorvid reversed the increase in total intracellular ROS induced by
   eNAMPT ([160]Fig. 5D). Resatorvid also inhibited eNAMPT-induced lipid
   peroxidation, as shown by the MDA levels and Liperfluo staining in
   AML12 cells ([161]Fig. 5E–F). Resatorvid treatment also inhibited
   eNAMPT-induced GSH depletion, reduced GPX4 expression, and decreased
   COX2 expression and cell death ([162]Fig. 5G–H). These data showed that
   eNAMPT-induced hepatocyte ferroptosis is mediated by the TLR4 receptor.
   Taken together, our data suggest that eNAMPT induces hepatocyte
   ferroptosis through TLR4-mitochondrial ROS-induced ferritinophagy.

Fig. 5.

   [163]Fig. 5
   [164]Open in a new tab

   Effects of a TLR4 inhibitor on eNAMPT-induced hepatocyte ferroptosis

   AML12 cells were incubated with recombinant NAMPT protein at 10 ng/mL
   and treated with or without Resatorvid (100 nM, a TLR4 inhibitor) for
   24 h. (A) Oxygen consumption rates (OCRs) were measured. (B)
   Representative immunofluorescence images of MitoSOX staining. (C) The
   expression of FTH1, LC3, and p62 was measured by Western blotting. The
   results are expressed as fold changes relative to the control. (D)
   Cellular Fe^2+ levels were measured in AML12 cells. Intracellular ROS
   levels were detected by the fluorescent probe DCFH-DA, and the
   fluorescence intensity was measured in AML12 cells. (E) Representative
   immunofluorescence images of Liperfluo staining. (F) The levels of MDA
   were measured in AML12 cells. (G) The expression of GPX4 and COX2 was
   measured via Western blotting. The results are expressed as fold
   changes relative to the control. (H) The GSH/GSSG ratio was measured in
   AML12 cells. Cell viability was measured in AML12 cells by a CCK8 kit.
   The data are expressed as the means ± SEMs from three independent
   experiments, statistical analysis by Two-way ANOVA. ^&p < 0.05 compared
   with the eNAMPT treatment group; ^#p < 0.05 compared with the DMSO
   group; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

2.7. Brown adipocyte-derived eNAMPT induces ferroptosis in hepatocytes

   Our results above revealed an increase in the level of eNAMPT upon
   ethanol consumption and its effects on liver function. However, the
   source of elevated eNAMPT is still unknown. Studies have shown that
   eNAMPT is secreted mainly from visceral and subcutaneous fat [[165]39].
   In addition, our previous study reported that ethanol intake can reduce
   the protein expression of NAMPT in the liver compared with control mice
   [[166]40]. In the present study, we found that the expression of NAMPT
   in WAT did not change in ethanol-fed mice. Interestingly, the
   expression of NAMPT was significantly increased in the BAT of
   ethanol-fed mice ([167]Fig. 6A). To better understand the effect of
   ethanol on eNAMPT secretion, we differentiated 3T3-L1 cells into mature
   white adipocytes and differentiated stromal vascular fraction cells
   (SVFs) into mature brown adipocytes. Mature brown adipocytes can
   secrete eNAMPT into media ([168]Fig. S4C). Our results showed that
   ethanol significantly increased NAMPT expression in brown adipocytes
   and promoted the secretion of eNAMPT, which was not observed in white
   adipocytes ([169]Fig. 6B). To investigate the role of adipocyte-derived
   eNAMPT in the regulation of ferroptosis in hepatocytes, we performed a
   coculture experiment. After treating brown adipocytes with ethanol, the
   conditioned medium (CM) was collected to culture AML12 cells ([170]Fig.
   6C) or HepG2 cells ([171]Fig. S3F). The CM derived from ethanol-treated
   brown adipocytes (CM-ethanol) had a much greater level of eNAMPT, which
   was significantly removed by immunoprecipitation ([172]Fig. 6D).
   CM-ethanol induces ferritinophagy, iron accumulation and lipid
   peroxidation in AML12 cells ([173]Fig. 6E–F). Similarly, after
   CM-ethanol incubation, the GSH/GSSG ratio and GPX4 expression
   decreased, while the COX2 expression increased in AML12 cells
   ([174]Fig. 6G–H). However, when eNAMPT was removed from CM-ethanol,
   these effects were significantly inhibited, ultimately reversing the
   death of AML12 cells ([175]Fig. 6H). These results indicate that brown
   adipocyte-derived eNAMPT is a key factor for ferroptosis in ALD.

Fig. 6.

   [176]Fig. 6
   [177]Open in a new tab

   Brown adipocyte-derived eNAMPT induces ferroptosis in hepatocytes

   (A) Mice were treated with chronic-plus-binge ethanol for 10 days, the
   expression of the NAMPT protein in mouse BAT and WAT was measured by
   Western blot, and the results are expressed as fold changes relative to
   the control, statistical analysis by Student's t-test. (B) Stromal
   vascular fraction cells (SVFs) were isolated and differentiated into
   mature brown adipocytes, and 3T3-L1 cells were differentiated into
   mature white adipocytes and then treated with or without EtOH
   (100 mmol/L) for 96 h. The protein expression of NAMPT in adipocytes
   and the levels of eNAMPT in the medium were measured via western
   blotting. The results are expressed as fold changes relative to the
   control, statistical analysis by Student's t-test. (C–H) The eNAMPT in
   the media collected from brown adipocytes after ethanol intervention
   was removed by immunoprecipitation (C), statistical analysis by Two-way
   ANOVA. (D) eNAMPT levels in the conditioned medium (CM) of brown
   adipocytes were measured via ELISA. The CM was cultured with
   AML12 cells for 24 h. (E) The expression of FTH1, LC3, and p62 was
   measured by Western blotting. The results are expressed as fold changes
   relative to the control. (F) Cellular Fe^2+ levels were measured in
   AML12 cells. The levels of cellular MDA were measured in AML12 cells.
   (G) The expression of GPX4 and COX2 was measured via Western blotting.
   The results are expressed as fold changes relative to the control. (H)
   The GSH/GSSG ratio was measured in AML12 cells. Cell viability was
   measured in AML12 cells by a CCK8 kit. The data are expressed as the
   means ± SEMs from three independent experiments. ^&p < 0.05 compared
   with the EtOH + IgG group; ^#p < 0.05 compared with the CON + IgG
   group; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (For
   interpretation of the references to color in this figure legend, the