Leptin, a hormone predominantly derived from adipose tissue, has long been considered to play regulatory roles in food intake and energy expenditure, since the discovery of obese gene (Zhang et al., 1994). A growing body of recent evidence indicates that leptin possesses a variety of biological functions, in addition to the potent metabolic functions. In particular, accumulating evidence suggests the implication of leptin signaling in the development of liver diseases at multiple stages, including steatohepatitis, hepatic fibrosis, and cirrhosis (Chatterjee et al., 2013; Zhang et al., 2021). Interestingly, plasma leptin levels are markedly enhanced in the obese individuals and leptin signaling contributes to the development of liver diseases associated with obesity (Rotundo et al., 2018). Apart from the metabolic regulation during liver diseases, leptin directly exerts cytotoxic effects on liver cells. We have also previously shown that leptin treatment causes a decrease in viability of rat hepatocytes by activating NLRP3 inflammasomes (Baral and Park, 2021), which act as a signaling platform for activation of the innate immune system through maturation of interleukin family of pro-inflammatory cytokines, such as interleukin-1β (IL-1β), IL-18, and IL-33 (Szabo and Csak, 2012). While leptin has been shown to exert direct cytotoxic effect in hepatocytes through NLRP3 inflammasomes, its underlying molecular mechanisms remain elusive.

IL-1β signaling plays important roles in the development and/or progression of many inflammation-associated diseases, such as arthritis and diabetes mellitus (Mirea et al., 2018). Additionally, there is a growing appreciation that IL-1β signaling is implicated in various instances of hepatic damage, including drug-, ischemia reperfusion-, radiation-, and ethanol-induced liver injury (Petrasek et al., 2012). There has been increasing recent evidence demonstrating that IL-1β is also involved in cell fate decisions in a complicated manner. For example, although IL-1β signaling was originally reported to induce pyroptotic and apoptotic cell death in various types of cells (Giannoukakis et al., 1999; Lebeaupin et al., 2015; Baral and Park, 2021), recent studies have revealed that it also potentially leads to growth of cancer cells (Raut et al., 2019), implying that the exact role of IL-1β signaling must be determined in a context-dependent manner. While IL-1β is mainly expressed in immune cells in liver, it is also produced by parenchymal cells. There is a growing appreciation that IL-1β maturation in hepatocytes via inflammasomes activation contributes to various types of liver cell death (Szabo and Csak, 2012; Baral and Park, 2021), although the underlying mechanisms have not been clarified.

Gene expression and maturation of IL-1β is tightly regulated at multiple steps. IL-1β is initially synthesized as an inactive precursor and converted into a biologically active form through proteolytic cleavages (Martinon et al., 2002). Maturation of IL-1β is mediated via inflammasomes activation. In this process, upon exposure to various types of stimuli, oligomerization of NLRP3 inflammasomes components leads to activation of caspase-1, resulting in the cleavage and maturation of IL-1β. Mature IL-1β is then secreted into the extracellular milieu via multiple mechanisms, including autophagy, shedding of microvesicles, and pyroptosis (Lopez-Castejon and Brough, 2011). The biological actions of IL-1β are initiated by binding with its receptor (IL-1R1), which causes conformational change in the receptor, facilitating receptor complex formation and leading to the modulation of various downstream signaling pathways, including p38MAPK (Kulawik et al., 2017) and AKT (Nov et al., 2010). AKT and p38MAPK signaling have been shown to play important roles in cell fate decision. Specifically, activation of p38 MAPK leads to apoptotic-, pyroptotic-, and necrotic cell death (Saldeen et al., 2001; Wang et al., 2005; Zhou et al., 2019), while its inactivation contributes to liver regeneration after partial hepatectomy (Campbell et al., 2011). AKT signaling has also received attention as a modulator of cell death/survival and the cell cycle. AKT activation leads to cell survival and inhibits apoptosis in liver cells (Jing et al., 2019), whereas dephosphorylation induces cell death (Schulze-Bergkamen et al., 2004).

Cell cycle, a series of events involved in cell growth and division, is regulated by a number of regulatory proteins in a concerted action. Indeed, a group of cyclins and cyclin-dependent kinases (CDKs) promote the progression of the cycle, while various CDK inhibitors, including p53, p21, p16, and retinoblastoma, act as negative regulators of the cell cycle (Kumari and Jat, 2021). It has been well documented that leptin induces cell cycle progression in cancer cells via up-regulation of cyclin D1 (Chen et al., 2007), regulation of ubiquitin specific protease 2 (Nepal et al., 2015), and inhibition of p21WAF1/CIP1 expression (Ptak et al., 2013), demonstrating its oncogenic actions. In contrast, increasing recent evidence indicate that leptin induces cell cycle arrest and senescence in non-cancerous cells, including chondrocyte (Zhang et al., 2016), neuronal cells (Segura et al., 2015), mesenchymal stem cells (Chen et al., 2015), and chondrogenic progenitor cells (Zhao et al., 2016). Based on previous reports, although a potential link between leptin and cell cycle regulation has been suggested, its effects and precise mechanisms vary depending on the context.

Leptin exerts a direct cytotoxic effect in hepatocytes, which may be critical in the development of pathological conditions in the liver. In the present study, to better understand the mechanisms underlying leptin-induced hepatic damage, we elucidated the molecular mechanisms by which leptin induces cytotoxic effects in hepatocytes, particularly focusing on the role of IL-1β signaling. We have demonstrated that maturation and secretion of IL-1β critically contributes to the apoptotic cell death and cell cycle arrest by leptin in rat hepatocytes. In addition, cell death and cell cycle arrest by IL-1β signaling are mediated, at least in part, via activation of p38MAPK and suppression of AKT signaling.

Materials

Type IV collagenase (C5138), type I collagen (C3867), Ac-YVAD-cmk (SML0429), a pharmacological inhibitor of caspase-1, Hank’s Balanced Salt Solution (HBSS) (H4891), and recombinant leptin (L3772) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Propidium iodide dye (ab139418), MitoOrange dye (ab138898), and antibodies against cytokeratin-18 (ab181597) and p16 (ab51243) were procured from Abcam (Cambridge, UK). SC79 (HY-18749) were obtained from MedChem Express (Monmouth Junction, NJ, USA). Annexin V & 7-AAD assay kit (640922) was purchased from BioLegend (San Diego, CA, USA). Primary antibodies against Bax (2772), Bcl-2 (2876), pro-caspase-3 (9662), cleaved caspase-3 (9664), PARP (9542), phosphor-AKT (4060S), phosphor-GSK3β (5558S), and phosphor-p38MAPK (9215) were acquired from Cell Signaling Technologies (Danvers, CO, USA). IL-1β (MAB501) and IgG1 isotype control (MAB002) antibodies were bought from R&D Systems (Minneapolis, MN, USA). Anti-rabbit (31460) and anti-mouse (31430) horseradish-conjugated secondary antibodies were purchased from Thermo Scientific (Waltham, MA, USA).

Isolation and culture of rat hepatocytes

Hepatocytes were isolated from 6-7 weeks old male Sprague Dawley (SD) rats using a two-step collagenase perfusion process as described previously (Baral and Park, 2021). All animal experiments were performed as per the guidelines of Yeungnam University Institutional Animal Care and Use of Committee (IACUC). The experimental protocols were reviewed and approved by the Yeungnam University IACUC (Approval number: 2022-016). Briefly, after cannulation of hepatic portal vein using an 18G catheter, the liver was perfused with HBSS free of Ca²⁺ and Mg²⁺, and type IV collagenase (0.05%). Collagenase perfusion was initiated once the liver turned pale showing clear signs of RBC removal. After digestion of the liver, cells were collected by filtering through a cell strainer (100 µm) and washed twice with HBSS. Cells were resuspended in William’s Medium E containing 10% fetal bovine serum and 1% penicillin/streptomycin and seeded in collagen-coated dishes. After 2 h, the media were changed to remove the unattached cells.

Generation of IL-1R1 conditional knock-out (IL-1R1 KO hepatocyte) mice and in vivo experiments

IL-1R1^loxP/loxP mice (# 028398) and mice expressing Cre transgene driven by the albumin promoter (Alb^Cre mice) (# 003574) on a C57BL/6 background were purchased from Jackson Laboratories (Farmington, CT, USA) and were housed under normal-pathogen free conditions. IL-1R^1loxP/loxP and ALb^Cre mice were bred to obtain IL-1R^1fl/fl: Alb^Cre (hereafter referred to as IL-1R1 KO mice). Similarly, age- and sex-matched littermates with IL-1R1^fl/fl but not expressing Cre were used as wild type controls. Mice were then grouped based on their genetic backgrounds. Male mice aged between 6 to 8 weeks were used for the experiments.

Expression levels of IL-1R1 and Cre in WT and IL-1R1 KO mice were determined using PCR amplification. After incubating the tail piece in alkaline lysis buffer, the DNA samples were amplified using DreamTaq DNA polymerase system (Thermo Scientific). PCR products were subjected to gel electrophoresis (1.5-3% agarose gel), and images were captured using a Vilber Fusion Solo system (Vilber, Collegien, France).

Mice were administered leptin (1 mg/kg) twice daily for 15 days by intraperitoneal injection, whereas control group mice received an equivalent volume of 1X PBS via the same route. At the end of the treatment, animals were sacrificed by anesthetization, and the livers were excised and subjected for further experiments. For western blot analysis, the tissues were homogenized using radioimmunoprecipitation assay (RIPA) lysis buffer containing 1% protease inhibitors cocktail and lysed further using sonicator (Sonics & Materials Inc, Newtown, CT, USA).

Measurement of cell viability (MTS assay)

Cell viability was measured based on the conversion of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium (MTS) solution into formazan product using the Cell Titer 96 Aqueous One kit (Promega Cooperation, Madison, WI, USA), as described previously (Lee et al., 2022). Briefly, rat hepatocytes were seeded into collagen-coated 96-well plates at a density of 5×10⁴ cells/well. After overnight culture and treatment with leptin and the agents indicated, cells were incubated with MTS reagent (20 μL) for 2 h. Cell viability was determined by measuring the absorbance at 490 nm using SPECTROstar Nano microplate reader (BMG LABTECH, Allmendgrün, Germany).

Western blot analysis

For measuring the expression levels of the genes of interest, Western blot analysis was carried out as described previously (Nguyen et al., 2023). Briefly, hepatocytes were seeded in collagen-coated 35-mm dishes at a density of 1×10⁶ cells/dish. After treatment as indicated in the figure legends, cells were extracted with RIPA lysis buffer containing protease and phosphatase inhibitors cocktail. After centrifugation at 12,000 g for 10 min, the supernatants were used for western blot analysis. Equal amounts of proteins (30-40 μg) were loaded onto sodium dodecyl-sulfate polyacrylamide gel and separated by electrophoresis (SDS-PAGE). For the measurement of secreted IL-1β, trichloroacetic acid (TCA) protein precipitation assay was performed as described previously (Khakurel and Park, 2018). In brief, cell culture media were collected and the proteins in the media were precipitated with TCA at 4°C. The pellets were then washed with cold acetone and subjected to SDS-PAGE. Proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was incubated with 5% skim milk for blocking followed by incubation with a primary antibody and a respective secondary antibody. The resultant immunocomplexes were finally detected by LAS 4000 mini (Fujifilm, Tokyo, Japan) using enhanced chemiluminescence (ECL).

Preparation of conditioned media

Conditioned media were prepared as described previously (Pun et al., 2015). Briefly, after treatments with leptin and/or inhibitors as indicated, the cell culture media were collected as conditioned media (Akbal et al., 2022) and filtered through 0.2 µM filter. The CM were mixed with fresh media at a 1:2 ratio and used to treat a fresh set of hepatocytes as indicated in the figure legends.

Annexin V/7-AAD binding assay

Apoptosis of hepatocytes were monitored using Annexin V Apoptosis Detection Kit (BioLegend) according to the manufacturer’s instruction. Briefly, after treatment as indicated, cells were detached from the culture plate, resuspended in Annexin V Binding Buffer, and incubated with staining buffer containing FITC-Annexin V and 7-AAD for 15 min in the dark at room temperature. Finally, apoptotic cells were detected by flow cytometry analysis using BD FACS Verse (BD Biosciences, Franklin lakes, NJ, USA).

Cell cycle analysis

Distribution of the cells in each cell cycle phase was monitored by propidium iodide (PI) staining and flow cytometry analysis as described previously (Pham et al., 2023). In brief, after indicated treatments, cells were collected, washed, fixed with 70% cold ethanol, and stained with PI for 30 min in the presence of an RNase I inhibitor. Cell cycle distribution was monitored by flow cytometry analysis using a BD FACS Verse (BD Biosciences). Percentage of the cells in each phase was calculated using FlowJo 7.6 software (Ashland, OR, USA).

Measurement of mitochondrial membrane potential

Mitochondrial membrane potential in hepatocytes was analyzed by staining with MitoOrange dye according to the manufacturer’s instructions. Briefly, cells were detached, incubated with MitoOrange Dye (2 µL, 200 ×) for 30 min at 37°C, and suspended in the assay buffer. Mitochondrial membrane potential was monitored by flow cytometry analysis.

Transient transfection with small interfering RNA (siRNA)

For gene silencing of the gene of interest, hepatocytes were seeded in a collagen-coated 35-mm dish at a density of 5×10⁵ cells/dish. After overnight culture, cells were transfected with a siRNA targeting the gene of interest or scrambled control siRNA using Lipofectamine RNAi MAX (Thermo Fisher, Rockford, IL, USA) according to the manufacturer’s instructions. Gene silencing efficiency was monitored by western blot analysis after 24 h after transfection. The oligonucleotide siRNAs were purchased from Bioneer (Daejon, Korea). The siRNA sequences used are presented in Table 1.

Table 1 Sequences of the nucleotides used for siRNA tranfection

Target gene	Forward sequence	Reverse sequence
IL-1R1	5'-CUAUGAUGCCUAUGUUCUU-3'	5'-AAGAACAUAGGCAUCAUAG-3'
P16	5'-GAUGAUGGGCAACGUCAAA-3'	5'-CAACGCGAGACUAGCAUAU-3'
Scrambled siRNA	5'-CCUACGCCACCAAUUUCGU-3'	5'-ACGAAAUUGGUGGCGUAGG-3'

RNA isolation and real time-quantitative polymerase chain reaction (RT-qPCR)

The mRNA levels of the target genes were determined by RT-qPCR as described previously (Pham and Park, 2022). In brief, hepatocytes were seeded in a collagen-coated 35-mm dish at a density of 1×10⁶ cells/dish. Total cellular RNA was extracted using Qiazol lysis reagent (Qiagen, Hilden, Germany). Total RNA (1 µg) was used for the synthesis of complementary DNA (cDNA), which was subsequently amplified by a Light Cycler 1.5 system (Roche Diagnostics, Mannheim, Germany) using an absolute SYBR green capillary mix (Thermo Scientific). The sequences of the primers used in this study are listed in Table 2.

Table 2 Sequences of the primers used for PCR amplification

Target gene	Forward sequence	Reverse sequence
p16	5'-CGGTATCTACTCTCCTCCGC-3'	5'-GTTGCCAGAAGTGAAGCCAA-3'
p21	5'-AACTGGGGAGGGCTTTCTTT-3'	5'-TGTCTTGTCTTCGCTGAGGT-3'
p27	5'-AACTAACCCGGGACTTGGAG-3'	5'-GAGACCCAATTGAAGGCACC-3'
GAPDH	5'-TGAACGGGGAAGCTCACTGG-3'	5'-TCCACCACCCTGTTGCTGTA-3'
Cyclin D1	5'-CCCCAACAACTTCCTCTCCT-3'	5'-AGCTTCTTCCTCCACTTCCC-3'
Cyclin E1	5'-AAGGAGGGTGCTACTTGACC-3'	5'-TCAGCTGACAGTGGAGAAGG-3'

Statistical analysis

Values are presented as mean ± standard error of the mean (SEM) from at least three independent experiments. Data were analyzed by One-Way-Analysis of Variance with Tukey’s multiple comparison test using GraphPad Prism version 5.0 (La Jolla, CA, USA). Groups were considered statistically significant if p<0.05.

IL-1β signaling plays a critical role in leptin-induced cell cycle arrest via upregulation of p16 in rat hepatocytes

We have previously shown the involvement of IL-1β signaling in cytotoxic effect of leptin in hepatocytes. In this study, we have further elucidated the molecular mechanisms by which IL-1β signaling mediates decreased viability of hepatocytes. To this end, we first confirmed the effect of leptin on activation and secretion of IL-1β in our experimental condition. As shown in Fig. 1A, leptin significantly enhances secretion of mature IL-1β in a time-dependent manner, as determined by western blot analysis using TCA-precipitates of the culture media, which is in line with the previous reports demonstrating the intracellular activation of IL-1β by leptin (Baral and Park, 2021). Given that cell viability is determined by the rate of cell death and proliferation, we first investigated whether leptin and IL-1β signaling modulate the cell cycle in hepatocytes. As shown in Fig. 1, leptin treatment induced significant increases in proportions of cells in the sub G1 and G1 phase, but decreased those in the S and G2 phase (Fig. 1B, 1C). Interestingly, pretreatment with interleukin-1 receptor antagonist (IL-1Ra) caused restoration of typical proportions in each cell cycle phase (Fig. 1B). Essentially similar results were observed by gene silencing of IL-1 receptor-1 (IL-1R1) (Fig. 1C), suggesting that leptin-induced cell cycle arrest is mediated via IL-1β signaling. To further characterize leptin-induced cell cycle arrest, we measured the expression levels of cell cycle regulators and observed that leptin significantly upregulates p16 expression at both mRNA and protein levels (Fig. 1D-1F), whereas no significant effects were observed in other cell cycle regulators, including p21, p27, cyclin D1, and cyclin E1 (Supplementary Fig. 1). In subsequent experiments to verify the functional role of p16 in leptin-modulation of the cell cycle, gene silencing of p16 restored changes in cell populations in each cell cycle phase (Fig. 1G). In addition, pretreatment with IL-1Ra notably inhibited leptin-induced p16 expression at both mRNA and protein levels (Fig. 1H, 1I, respectively), indicating that IL-1β signaling is implicated in leptin-induced upregulation of p16. Since IL-1β is activated through NLRP3 inflammasomes, we also investigated the role of NLRP3 inflammasomes and found that treatment with NLRP3 inflammasomes inhibitors (MCC950 and Ac-YVAD-cmk) almost completely abolished leptin-stimulated p16 expression (Fig. 1J), which are similar to the results from IL-1Ra pretreatment. Moreover, treatment with conditioned media obtained from leptin-treated hepatocytes cultures significantly increased p16 expression, which was restored in the presence of Ac-YVAD-cmk (Fig. 1K). Collectively, these results suggest that cell cycle arrest by leptin is mediated through IL-1β signaling, at least in part, via upregulation of p16.

Figure 1. Critical role of IL-1β signaling in leptin-induced cell cycle arrest in rat hepatocytes. (A) Hepatocytes were isolated from rats as indicated in the methods and treated with leptin (250 ng/mL) for the indicated time periods. The amount of mature IL-1β secreted into the media were determined by western blot analysis using TCA-precipitated protein as described in methods. Loading of the equal amounts of proteins was confirmed by Ponceau red staining. (B, C) Rat hepatocytes were pretreated with IL-1Ra (100 ng/mL) for 1 h (B), or transfected with siRNA (100 nM) targeting IL-1R1 or scrambled control siRNA for 24 h (C), followed by treatment with leptin (250 ng/mL) for additional 24 h. Cells were stained with propidium iodide (PI) and cell cycle analysis was carried out by flow cytometry. Representative images from three independent experiments are presented. The percentage of the cells distributed in each cell cycle phase from three experiments is presented in the right panel. (D) Cells were treated with leptin (250 ng/mL) for the indicated time points. Messenger RNA level of p16 was determined by qRT-PCR using GAPDH as an internal control. (E, F) Hepatocytes were treated with leptin (250 ng/mL) for the indicated time durations (E) or treated with different doses of leptin for 8 h (F). Protein expression level of p16 was determined by western blot analysis. (G) Cells were transfected with p16 siRNA (100 nM) for 24 h followed by treatment with leptin (250 ng/mL) for additional 24 h. Cell cycle analysis was performed by flow cytometry. Representative images from three independent experiments are presented. The percentage of the cells in each cell cycle phase from three experiments is displayed in the right panel. Gene silencing efficiency was monitored by western blot analysis after 24 h of transfection and presented in the upper panel. (H) Hepatocytes were treated with leptin (250 ng/mL) for 6 h in the absence or presence of IL-1Ra (100 ng/mL). p16 mRNA level was determined by qRT-PCR. (I, J) Hepatocytes were pretreated with IL-1Ra (100 ng/mL) (I), or MCC950 (1 µM) or Ac-YVAD-cmk (1 µM) (J) for 1 h followed by incubation with leptin (250 ng/mL) for 8 h. Protein expression levels of p16 were measured by western blot analysis. (K) Hepatocytes were treated with conditioned media obtained from hepatocytes culture treated with leptin in the absence or presence of Ac-YVAD-cmk for 8 h. Expression level of p16 was measured by western blot analysis. β-actin was used as an internal control. In the western blot analyses, the band intensities were analyzed by densitometric analysis and are presented in the lower panel of the images. Values are presented as mean ± SEM, n=3. *p<0.05 compared with control cells. ^#p<0.05 in comparison to the cells treated with leptin.

IL-1β signaling contributes to apoptotic cell death by leptin in rat hepatocytes

To further characterize the decreased viability of hepatocytes caused by leptin, we examined whether IL-1β secretion also contributes to apoptotic cell death. As shown in Fig. 2, leptin treatment prominently enhanced apoptosis in hepatocytes, as evidenced by annexin V/7-AAD binding assay. Interestingly, treatment with IL-1Ra and gene silencing of IL-1R1 significantly alleviated leptin-induced apoptotic cell death (Fig. 2A, 2B, respectively). Additionally, neutralization of IL-1β by treatment with IL-1β monoclonal antibody caused restoration of leptin-decreased hepatocyte viability (Supplementary Fig. 2), collectively suggesting the critical role of IL-1β signaling in leptin-induced apoptosis of hepatocytes. The role of IL-1β signaling was further confirmed by measuring the levels of the genes related with apoptosis. As indicated in Fig. 2, leptin-induced cleavage of cytokeratin-18, a critical apoptotic marker in hepatocytes, was markedly prevented by pretreatment with IL-1Ra (Fig. 2C) and silencing of IL-1R1 (Fig. 2D). In addition, pretreatment with IL-1Ra also substantially suppressed leptin-induced increases in other conventional apoptotic markers, including cleaved PARP (Fig. 2E) and Bax (Fig. 2F), while recovered the suppressed Bcl-2 expression (Fig. 2G). Furthermore, treatment with conditioned media obtained from hepatocyte culture in the presence of Ac-YVAD-cmk showed a marked reduction in leptin-stimulated caspase-3 cleavage (Fig. 2H). Finally, leptin-induced reduction in mitochondrial membrane potential, which can promote the release of apoptosis promoters such as cytochrome c, apoptosis-inducing factors (AIFs), and mitochondrial DNA, was restored to the almost normal levels by IL-1Ra pretreatment (Fig. 2I). Taken together, these results imply that IL-1β signaling plays a critical role in leptin-induced apoptosis of hepatocytes.

Figure 2. Implication of IL-1β signaling in apoptotic cell death by leptin in rat hepatocytes. (A, B) Rat hepatocytes were pretreated with IL-1Ra (100 ng/mL) for 1 h (A), or cells were transfected with siRNA targeting IL-1R1 or scrambled control siRNA (100 nM) (B), followed by treatment with leptin (250 ng/mL) for additional 24 h. Apoptotic cell death was determined by annexin V binding assay. Representative images from three independent experiments are presented. Cells in lower right quadrant indicate early apoptotic cells and upper right quadrant indicate late apoptotic cells. The percentage of cells in each quadrant from three independent experiments were quantified and presented in the right panel of this assay. (C, D) Cells were treated with IL-1Ra (100 ng/mL) for 1 h (C), or transfected with siRNA targeting IL-1R1 or scrambled control siRNA (100 nM) (D), followed by treatment with leptin (250 ng/mL) for additional 8 h. Expression levels of total and cleaved cytokeratin-18 were measured by western blot analysis. (E-G) Cells were treated with leptin (250 ng/mL) for 8 h in the absence or presence of IL-1Ra (100 ng/mL). Protein expression levels of (cleaved) PARP (E), Bax (F), and Bcl-2 (G) were measured by western blot analysis. (H) Conditioned media obtained from hepatocytes culture treated with leptin in the absence or presence of Ac-YVAD-cmk were used for the treatment of a fresh set of hepatocytes (CM: fresh WME (1:2)). Expression levels of (cleaved) caspase-3 were analyzed by western blot analysis. (I) Rat hepatocytes were treated with leptin (250 ng/mL) for 8 h in the absence or presence of IL-1Ra (100 ng/mL). Cells were incubated with MitoOrange dye for 30 min and mitochondrial membrane potential was determined by flow cytometry analysis. In the western blot analyses, β-actin or total form of respective protein was used as an internal control. Statistical analysis for the band intensity determined by densitometric analysis are presented in the lower panel. Values are presented as fold change in comparison to control group and expressed as mean ± SEM, n=3. *p<0.05 compared to control cells. ^#p<0.05 in comparison to the cells treated with leptin.

Apoptosis and cell cycle arrest by IL-1β signaling is mediated via AKT inactivation in hepatocytes stimulated with leptin

We further elucidated the detailed molecular mechanisms by which IL-1β signaling mediates apoptosis and cell cycle arrest. Given that AKT pathway plays a critical role in the survival of hepatocytes (Jing et al., 2019), we examined whether regulation of AKT signaling is implicated in leptin-induced apoptosis and cell cycle arrest. To this end, we first assessed the effect of leptin on AKT phosphorylation and observed that leptin treatment rapidly induces dephosphorylation of AKT at Ser₄₇₃, which is returned to near normal levels at long time exposure (Fig. 3A). Dephosphorylation of AKT by leptin was restored by pretreatment with IL-1Ra (Fig. 3B) and inhibitors of NLRP3 inflammasomes (Fig. 3C). Moreover, dephosphorylation of AKT by exposure to the conditioned media obtained from hepatocyte culture treated with leptin was returned to the normal level in the presence of caspase-1 inhibitor (Fig. 3D), collectively suggesting that IL-1β signaling mediates dephosphorylation of AKT upon leptin exposure.

Figure 3. Mediating role of AKT dephosphorylation in apoptosis and cell cycle arrest by leptin in hepatocytes. (A) Hepatocytes were isolated and treated with leptin (250 ng/mL) for the given time periods. Phosphorylated AKT levels were measured by western blot analysis. (B, C) Hepatocytes were pretreated with IL-1Ra (B), or MCC950 or Ac-YVAD (C) for 1 h, followed by treatment with leptin (250 ng/mL) for 8 h. The levels of phosphorylated AKT were measured by western blot analysis. (D) Conditioned media obtained from hepatocyte culture treated with leptin in the absence or presence of Ac-YVAD-cmk were prepared as in methods. A fresh set of hepatocytes were treated with CM for 8 h. Phosphorylated AKT levels were determined by western blot analysis. (E-H) Hepatocytes were pretreated with SC79 (4 µg/mL) for 1 h followed by leptin stimulation for 24 h (E, G) or 8 h (F, H). (E) Cells were stained with annexin V/7-AAD and subjected to flow cytometry analysis for the detection of apoptotic cell death. Cells in lower right quadrant indicate early apoptosis and upper right quadrant indicate late apoptosis. Cells in each quadrant were quantified and presented in the right panel. (F) Expression levels of cleaved caspase-3 were determined by western blot analysis. (G) Cell cycle analysis was carried out by flow cytometry. Representative images from three independent experiments are presented. The percentage of the cells distributed in each cell cycle phase from three experiments is presented in the lower panel. (H) Expression levels of p16 was measured by western blot analysis. In the western blot analyses, β-actin was used as internal control. Band intensities were analyzed by densitometric analysis and are presented in the lower panel of the images. Values are presented as mean ± SEM, n=3. * denotes p<0.05 compared with control cells. ^# denotes p<0.05 in comparison to the cells treated with leptin.

In ensuing experiments to verify the functional role of AKT inactivation in leptin-induced hepatocytes death, leptin-stimulated annexin V/7-AAD binding was markedly restored by treatment with a pharmacological activator of AKT (SC79) (Fig. 3E). The formation of cleaved caspase-3 was also significantly suppressed by treatment with SC79 (Fig. 3F), indicating that AKT inactivation is implicated in leptin-induced apoptosis of hepatocyte. We further found that treatment with AKT activator reverted the changes in cell populations in each cell cycle phase (Fig. 3G) and suppression of p16 induction (Fig. 3H), implying that AKT inactivation also plays an important role in leptin-induced cell cycle arrest in hepatocytes.

Apoptosis and cell cycle arrest by AKT dephosphorylation is mediated via activation of GSK3β in rat hepatocytes

Upon further exploration, we found that leptin treatment activated GSK3β, which is known as modulator of apoptosis in hepatocytes (Chen et al., 2012) and considered a downstream target of AKT. GSK3β activity is determined by its phosphorylation status at ser9. As shown in Fig. 4, leptin treatment caused significant dephosphorylation of GSK3β, reflecting activation of GSK3β. In addition, as expected, activation (dephosphorylation) of GSK3β was prevented by pretreatment with AKT activator (Fig. 4A) and IL-1Ra (Fig. 4B). We further investigated whether GSK3β activation plays a role in leptin-induced apoptosis of hepatocyte. As shown in Fig. 4, inhibition of GSK3β by treatment with SB216763 significantly suppressed leptin-induced apoptosis as determined by annexin V/7-AAD staining (Fig. 4C) and measurement of caspase-3 cleavage (Fig. 4D). Moreover, cell cycle arrest by leptin was also mitigated upon inhibition of GSK3β along with restoration of upregulated p16 expression (Fig. 4E, 4F, respectively). Taken together, IL-1β signaling-mediated AKT inactivation and GSK3β activation critically contributes to leptin-induced apoptosis and cell cycle arrest in hepatocytes.

Figure 4. Role of GSK3β signaling in mediating apoptosis and cell cycle arrest by AKT dephosphorylation in hepatocytes treated with leptin. (A, B) Hepatocytes were pretreated with SC79 (4 µg/mL) (A) or IL-1Ra (100 ng/mL) (B) for 1 h followed by leptin (250 ng/mL) for additional 8 h. The levels of phosphor-GSK3β were measured by western blot analysis. (C-F) Hepatocytes were pretreated with SB216736 (5 µM), a pharmacological inhibitor of GSK3β, for 1 h followed by treatment with leptin (250 ng/mL) for 24 h (C, E) or 8 h (D, F). (C) Apoptotic cell death was detected by annexin V/7-AAD binding and flow cytometry analysis. Cell in each quadrant were quantified and presented in the right panel. (D) Expression levels of cleaved caspase-3 were measured by western blot analysis. (E) Cell cycle analysis was performed by PI staining and flow cytometry analysis. Percentage of the cells in each cell cycle phase is presented in the right panel. (F) Expression levels of p16 measured by western blot analysis. In both flow cytometry and western blot analyses, representative images from three independent experiments are shown. Values are presented as mean ± SEM, n=3. *p<0.05 compared with control cells, ^#p<0.05 in comparison to the cells treated with leptin.

p38MAPK activation contributes to IL-1β signaling-mediated apoptosis and cell cycle arrest in hepatocytes stimulated with leptin

p38MAPK, a typical stress-activated serine/threonine kinase, is considered a critical signaling molecule that modulates apoptosis and cell cycle arrest in hepatocytes (Awad et al., 2000; Jeon et al., 2014). Therefore, we further examined whether p38MAPK signaling is involved in the cytotoxic effect of leptin in hepatocytes. Leptin induces phosphorylation of p38MAPK in a time-dependent manner (Fig. 5A). In addition, pretreatment with SB203580, a pharmacological inhibitor of p38MAPK, significantly suppressed leptin-induced apoptosis, as assessed by annexin V and 7-AAD staining (Fig. 5B) and downregulated cleavage of caspase-3 (Fig. 5C). Moreover, leptin-induced cell cycle arrest and p16 upregulation were also substantially inhibited by pretreatment with SB203580 (Fig. 5D, 5E). Collectively, these results suggest that p38MAPK activation plays a role in apoptosis and cell cycle arrest by leptin. In ensuing experiments to elucidate the mechanisms leading to p38MAPK activation, we found that activation of AKT (through treatment with SC79) and inhibition of GSK3β (through treatment with SB216763), prevented p38MAPK phosphorylation (Fig. 5F, 5G, respectively). In addition, treatment with IL-1Ra also prominently suppressed leptin-induced p38MAPK activation (Fig. 5H). Together, p38MAPK activation by leptin is mediated via AKT inactivation and GSK3β activation.

Figure 5. Role of p38MAPK activation in apoptosis and cell cycle arrest by leptin in rat hepatocytes. (A) Hepatocytes were treated with leptin (250 ng/mL) for the indicated time and phosphor-p38MAPK levels were analyzed by western blot analysis. (B-E) Hepatocytes were pretreated with SB203580 (10 µM), a pharmacological inhibitor of p38MAPK, for 1 h followed by incubation with leptin (250 ng/mL) for additional 24 h (B, D) or 8 h (C, E). (B) Cells were stained with annexin V/7-AAD and apoptotic cell death was determined by flow cytometry analysis. Cells in each quadrant were quantified and presented in the right panel. (C) Expression levels of cleaved caspase-3 were measured by western blot analysis. (D) Cell cycle analysis was performed by PI staining and flow cytometry analysis. Cells in each phase of cell cycle is quantified and presented in the right panel. (E) Expression levels of p16 were measured by western blot analysis. (F-H) Hepatocytes were pretreated with SC79 (4 µg/mL) (F), SB216763 (5 µM) (G), or IL-1Ra (100 ng/mL) (H) for 1 h followed by incubation with leptin (250 ng/mL) for additional 8 h. Phosphor-p38MAPK levels were determined by western blot analysis. Representative images from at least three independent sets of experiment is shown for both flow cytometry and western blot analysis. In western blot analysis, the band intensities of the genes of interest were quantified by densitometric analysis and presented in the lower panel. β-actin or total form of corresponding protein was used as internal control. Values are presented as mean ± SEM, n=3. *p<0.05 in comparison to control cells, ^#p<0.05 in comparison to the cells treated with leptin.

Apoptosis, cell cycle arrest, and modulation of AKT/p38MAPK by leptin in the liver are prevented in hepatocyte specific IL-1R1 knockout mice

IL-1β-dependent signaling is conveyed by binding with its functional receptor (IL-1R1). To validate the role of IL-1β signaling in leptin-modulation of liver physiology in vivo, we utilized hepatocyte-specific IL-1R1 knockout mice using the LoxP/Cre system with albumin promoter. Hepatocyte- specific IL-1R1 gene deletion was confirmed (Fig. 6A-6C). Effects of leptin on the expression of apoptotic genes were examined in wild type and knock out mice. As shown in Fig. 6, leptin-induced upregulation of cleaved caspase-3 (Fig. 6D) and cytokeratin-18 (Fig. 6E) observed in the liver of WT mice were prevented in those of IL-1R1 KO mice. Similarly, leptin-induced upregulation of Bax (Fig. 6F) and downregulation of Bcl-2 (Fig. 6G) were significantly inhibited in IL-1R1 KO mice. Collectively, these results suggest that leptin induces apoptosis in vivo in an IL-1R1 signaling-dependent manner. Additionally, upregulation of p16 by leptin was also inhibited in the livers of IL-1R1 KO mice (Fig. 6H). Finally, inactivation of AKT (Fig. 6I) and activation of p38MAPK (Fig. 6J) by leptin were also prominently suppressed in IL-1R1 KO liver. All the results are essentially similar to those from in vitro observations and validate the functional role of IL-1β signaling in the modulation of cell cycle arrest and apoptosis by leptin in vivo.

Figure 6. Effects of leptin on apoptosis, p16 expression, and modulation of AKT/p38MAPK pathways in hepatocyte specific IL-1R1 knockout mice liver. (A) Hepatocyte specific IL-1R1 knockout mice was generated using loxP/Cre system. Messenger RNA levels of the corresponding genes were meaured by PCR amplification as described in the methods. In left panel, lane-1: IL-1R1^fl/fl mice, lane-2: heterozygous for IL-1R1, lane-3: WT for IL-1R1, lane-4: IL-1R1^fl/fl mice. In right panel, lanes 1 and 2: Cre recombinase detected, lanes 3^rd and 4^th: no expression of Cre recombinase. (B, C) Expression levels of IL-1R1 in liver (B) and hepatocytes (C) were measured by western blot analysis. (D-I) Mice were adminstered with leptin (1 mg/kg) intraperitoneally twice a day for 15 days. After treatment, livers were prepared as indicated in the methods and expression levels of cleaved caspase-3 (D), cleaved cytokeratin-18 (E), Bax (F), Bcl-2 (G), p16 (H), phosphor-AKT (I), and phosphor-p38MAPK (J) were measured by western blot analysis. Representative images from three mice out of total five mice in each group is presented. Values are presented as mean ± SEM, n=5. * denotes p<0.05 in comparison to WT-control. ^# denotes p<0.05 in comparison to WT-leptin.

Leptin, a hormone primarily produced by the adipose tissue, was originally reported to play a crucial role in regulating energy balance. Recent studies have demonstrated that its receptors are present throughout the body and modulate a variety of physiological responses, apart from its classical metabolic actions. In particular, there is a growing appreciation that high plasma levels of leptin, termed hyperleptinemia, is closely associated with the development of liver diseases at multiple stages (Chitturi et al., 2002). We and other researchers have reported that leptin exerts cytotoxic effects in hepatocytes (Zhang et al., 2021). However, the detailed molecular mechanisms underlying this phenomenon have not yet been elucidated. In this study, we have demonstrated that IL-1β, which is activated and secreted upon NLRP3 inflammasomes activation, plays a critical role in the cytotoxic effects of leptin by inducing both apoptosis and cell cycle arrest. In addition, decreased viability of hepatocytes by IL-1β signaling is mediated, at least in part, via inactivation of AKT and activation of p38MAPK signaling.

Abnormal production of pro-inflammatory cytokines is closely associated with the initiation, development, and progression of various types of liver diseases. In most cases, pro-inflammatory cytokines in the liver originate from immune cells, such as Kupffer cells and other infiltrating immune cells (Luan et al., 2018). Production of inflammatory cytokines in response to various stimuli leads to paracrine interactions between hepatocytes and immune cells in a complicated manner, potentially inducing hepatocyte death under various experimental conditions (Tilg et al., 2011). In this study, we have examined the potential role of IL-1β signaling, derived from hepatocytes, but not from immune cells, in leptin-induced cytotoxicity in cultured rat hepatocytes. We observed that IL-1β signaling is critical for apoptosis of hepatocytes based on the collective assessment of the annexin-V/7-AAD staining, cleavages of caspase-3, and cytokeratin-18 (Fig. 2A-2D). Moreover, IL-1β signaling also crucially contributes to cell cycle arrest and p16 induction by leptin (Fig. 1), collectively indicating that hepatocyte-derived mature IL-1β upon NLRP3 inflammasomes activation leads to hepatocytes death. With regards to the activation of NLP3 inflammasomes, we have previously shown that ROS/ER stress axis is critical for leptin-induced NLRP3 inflammasomes activation (Baral and Park, 2021). These results warrant an attention that the generation of mature IL-1β from hepatocyte, other than Kupffer cells and other immune cells, play a significant role in the decision of hepatocyte fate. To the best of our knowledge, this is the first report to highlight the significance of autocrine IL-1β signaling in the cell cycle arrest and apoptosis of hepatocyte.

We have previously reported that leptin induces pyroptotic cell death in hepatocyte (Baral and Park, 2021). In this study, we also found that leptin induces apoptosis in cultured hepatocytes (Fig. 2). The results observed in the present study and previous reports imply that different types of cell death occur in hepatocytes upon exposure to leptin. Although apoptosis and pyroptosis are distinct types of programmed cell death, they are interconnected in certain conditions. Interestingly, inflammasomes activation has been shown to initially lead to pyroptotic cell death, accompanied by a release of inflammatory cytokines and pro-inflammatory cell death, followed by the apoptotic pathway (Wallace et al., 2022). Herein, we observed that pretreatment with various types of pharmacological inhibitors of pyroptosis, including betaine, punicalagin, and disulfiram, significantly suppressed leptin-induced expression of the genes related with apoptosis (Supplementary Fig. 3). Additionally, gene silencing of gasdermin D, a marker of pyroptosis, significantly inhibited leptin-induced cleavage of caspase-3 and cytokeratin-18 (Supplementary Fig. 3). While these results suggest that pyroptosis might occur prior to apoptosis and would further contribute to apoptosis in hepatocytes treated with leptin, given the complex crosstalk between apoptosis and pyroptosis, further studies dealing with the relationship between pyroptosis and apoptosis by leptin would be needed to gain better insights into the mechanisms underlying the cytotoxic effects of leptin in liver cells.

AKT signaling is well known to play a critical role in the survival of liver cells. Leptin has been widely shown to phosphorylate AKT and activate its downstream signaling. For instance, leptin induces phosphorylation of AKT in cancer cells, which results in activation of various downstream signaling, leading to cancer cell growth (Saxena et al., 2007). Additionally, leptin induces phosphorylation of AKT in muscle, adipocytes, and hepatic stellate cells, which contributes to the modulation of insulin signaling, glucose metabolism, and development of hepatic fibrosis. Contrary to this notion, herein we observed that leptin dephosphorylates AKT at serine 473 through an IL-1β signaling-dependent mechanism (Fig. 3). Although leptin has been widely shown to activate AKT, differential effects on AKT phosphorylation have also been reported. For example, leptin treatment did not cause significant effect on AKT phosphorylation in mouse hepatocytes (Huang et al., 2022). In addition, chronic exposure to leptin, termed chronic hyper-leptinemia, inhibits AKT phosphorylation in adipocytes, resulting in disruption of insulin signaling (Gupta et al., 2017) suggesting that effect of leptin on AKT activation would be determined in a context-dependent manner, challenging with origin of the cells, duration of treatment, and the dose of leptin used. To the best of knowledge, this is the first report demonstrating the inhibitory effect of leptin on AKT signaling in liver cells. Given that AKT signaling is involved in a number of physiological processes in liver, investigating the effect of leptin on AKT signaling in more detail and elucidating these mechanisms would be valuable for understanding the diverse hepatic pathologies induced by leptin.

In addition to the potent pro-inflammatory properties, IL-1β signaling has been shown to induce cell cycle arrest and apoptosis in various types of cells (Guadagno et al., 2015). Herein, we also observed that leptin induces cell cycle arrest through IL-1β signaling. In a series of experiments for characterization of cell cycle arrest upon leptin treatment, we observed that leptin induces a significant increase in p16 expression through IL-1β signaling without significant effects on other cell cycle regulators (Fig. 1, Supplementary Fig. 1). p16^INK4A inhibits activation of cyclin dependent kinase (CDK-4 and CDK-6), which is necessary for the phosphorylation of retinoblastoma gene product (pRb), resulting in dissociation from E2F1 complex to promote cell cycle progression. Silencing of p16 was sufficient to increase in DNA synthesis in primary rat hepatocytes (Harashima et al., 2013). In line with the previous studies, we observed that upregulation of p16 critically contributes to cell cycle arrest in rat hepatocytes (Fig. 1G). Previous studies have shown that TNF-α, which is secreted during cell cycle arrest, promotes apoptosis of the cells undergoing cell cycle arrest (Kim et al., 2020). Given that leptin induces expression of TNF-α in various cell types (Lee et al., 2014), it is possible that cell cycle arrest induced by IL-1β signaling induces secretion of TNFα, which further causes apoptotic cell death in hepatocytes. Additionally, TNFα has been shown to induce apoptosis in liver cells via p38 MAPK-dependent mechanisms. Hence, based on the results from the present study and previous reports, it appears that multiple complicated signaling mechanisms could be involved in the decision of cell fate by leptin in hepatocytes with a pivotal role of IL-1β in the overall process.

In conclusion, data presented in this study demonstrated that IL-1β maturation and secretion upon NLRP3 inflammasomes activation critically contributes to leptin-induced cell cycle arrest and apoptosis in hepatocytes. Furthermore, we have elucidated that cytotoxic effects by IL-1β signaling is mediated via differential regulation of AKT and p38MAPK (Fig. 7). Given that leptin signaling is implicated in various types of hepatic injury, in addition to direct hepatotoxic effect, IL-1β would be a potential target for the treatment of liver pathologies associated with leptin.

Figure 7. Proposed model for the role of interleukin-1β signaling in leptin-induced hepatocyte death. Leptin induces maturation of interleukin-1β (IL-1β) through NLRP3 inflammasomes signaling, which itself was found to be activated via ER stress-dependent mechanism in our previous report (Baral and Park, 2021). Once IL-1β is activated, it is secreted into the extracellular milleu, where it binds to the type-I interleukin-1 receptor (IL-1R1) and transmits the signal that induce apoptosis and cell cycle arrest in hepatocytes. Mechanistically, conformational change of IL-1R1 upon binding with IL-1β leads to inactivation of AKT, which plays a role in the survival and proliferation of hepatocyte. Inactivation of AKT causes dephosphorylation (at Ser 9) and subsequent activation of GSK3β, further resulting in activation of the stress kinase p38MAPK. Finally, p38MAPK signaling critically contributes to p16-dependent cell cycle arrest and apoptotic cell death. Illustration created with BioRender.com.

This work was supported by the Yeungnam University research grant in 2022. The authors thank the Core Research Support Center for Natural Products and Medical Materials (CRCNM) for the technical support regarding the confocal microscopic analysis.

The authors declare that there are no competing interests.

Park PH; designed the study, analyzed the data, edit/wrote the manuscript. Baral A; performed the experiments, analyzed the data, wrote the manuscript.