Honokiol Attenuates Lipotoxicity in Hepatocytes via Activating SIRT3-AMPK Mediated Lipophagy

Background: Non-alcoholic fatty liver disease (NAFLD) is characterized by ectopic accumulation of triglycerides in the liver. Emerging evidence has demonstrated that lipophagy regulates lipid mobilization and energy homeostasis in liver. Sirtuin 3 (SIRT3), a mitochondrial NAD + -dependent deacetylase, modulates the activities of several substrates involving in autophagy and energy metabolism. Honokiol (HK) is a natural lignan from the plants of Magnolia genus that exhibits potent liver protective property. Methods: AML12 was challenged with 500 μM palmitic acid and 250 μM oleic acid mixture solution to induce lipotoxicity. The expression of autophagy-related and AMP-activated protein kinase (AMPK) pathway proteins was evaluated by Western blotting and immunouorescence staining. Intracellular lipid accumulation was validated by Nile red staining. Molecular docking analysis was performed on AutoDock 4.2. Results: HK (5 and 10 μM) was found to attenuate lipid accumulation through promoting SIRT3-AMPK-mediated autophagy, mainly on lipid droplets. HK had hydrophobic interaction with amino acid residues (PHE294, GLU323 and VAL324) and NAD + . Moreover, HK improved mitochondrial function to enhance lipolysis, through decreasing the acetylated long-chain acyl-CoA dehydrogenase level. Conclusions: These results suggest that HK could ameliorate lipotoxicity in hepatocytes by activating SIRT3-AMPK-lipophagy axis, which might be a potential therapeutic agent against NAFLD.


Background
Non-alcoholic fatty liver disease (NAFLD) is prevailing in recent decades, which is closely related to nonalcoholic steatohepatitis (NASH), liver brosis and even hepatocellular carcinoma [1]. NAFLD is resulted from the ectopic accumulation of liver lipid, accompanied by lipotoxicity and subsequent metabolic abnormalities [2]. NAFLD occurs when the abnormal accumulation of triglycerides (TG) cannot compensate by the consumption [3]. Autophagy is a cellular recycling process that achieves energy homeostasis through lysosomal dependent degradation. The development of NAFLD is positively associated with impaired autophagy [4,5]. Lipophagy describes such a process that lipid droplets (LDs) are engulfed into autolysosomes, causing the release of free fatty acids (FFAs) [6]. This actually opens up the possibility to alleviate lipotoxicity in hepatocytes.

Cell viability
The viability of AML12 cells was determined by MTT as previously described [10]. The working solution of HK was prepared immediately before use through diluting the stock solution (10 mM in DMSO) with fresh complete medium.

Immunoblotting
Protein concentration was quanti ed with a BCA Protein Assay Kit after lysing the cells with RIPA lysis buffer (containing 1% protease inhibitor cocktail and 1% phenylmethane sulfonyl uoride). Proteins (20-30 μg) were separated using 5-12% SDS-PAGE and then transferred to PVDF membranes. The membranes were rstly blocked with 5% defatted milk for 2 h at room temperature, followed by overnight incubation of speci c primary antibodies at 4 °C and further incubation of secondary antibodies for 1 h at room temperature. SuperSignal West Femto Maximum Sensitivity Substrate kit was used to develop the signals. Visualization of the speci c protein bands were achieved on the ChemiDoc MP Imaging System, and the bands were quantitated with Image Lab 5.1 (Bio-Rad laboratories, Hercules, CA, USA).

RNA trans fection and adenovirus infection
Cells were transfected with 2 μg shRNA using Lipofectamine 3000 (Thermo-Fihser). After 6 h, cells were switched into fresh medium and incubated for a day. Then, cells were successively selected with puromycin (2 μg/mL) for 6 days and puromycin (4 μg/mL) for another 6 days before cells were pooled together.
Cells (2 × 10 5 ) were seeded in 6-well plates and infected with 10 μL Ad-mCherry-GFP-LC3 (multiplicity of infection = 5) using Lipofectamine 3000 after incubating for 24 h, followed by switch to fresh medium after 6 h and incubation for an additional 24 h. Then, cells were pooled together and ready for further investigations.

Confocal immuno uorescence microscopy
After seeding, cells were xed in formalin (10%), blocked with goat serum (2.5%), and incubated with primary antibodies as well as Texas Red-conjugated secondary antibodies. The nuclei were stained with DAPI. Leica TCS SP8 confocal uorescence microscope (Leica, Buffalo Grove, IL, USA) was used to capture the images.

Nile red staining
Nile red staining was conducted as previously reported [22]. Brie y, AML12 hepatocytes were xed with formaldehyde (10%) and stained with Nile red (1 μg/mL). After incubating for 30 min at 4 °C and washing with PBS, the stained LDs were observed with uorescence microscopy, and quantitated with ow cytometer with excitation and emission wavelength at 530 and 590 nm, respectively.
Determination of cellular triglycerides TG content in cell lysate was determined by using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) and normalized by protein concentration.

Molecular docking analysis
Docking was performed on AutoDock 4.2. The crystal structure of the quaternary complex (SIRT3, a substrate, NAD + , and the speci c agonist amiodarone hydrochloride; PDB ID: 5H4D) [23] was employed as the receptor. The protein was rstly prepared at pH 7.4 with all the water molecules removed and corresponding hydrogen atoms added. The 3D structure of HK was downloaded from the PubChem database. Gasteiger charge was calculated and AD4 atom type was assigned, and a 50 Å × 48 Å × 40 Å grid box with 375 Å spacing was placed to include the surface of the catalytic cleft with the assistance of amiodarone hydrochloride. The genetic searching algorithm was chosen for docking calculations, and 50 genetic algorithm runs were performed. Other parameters were set as default. The acquired poses were clustered with a tolerance of 2.0 Å.

Lipid droplets isolation
LDs were isolated from AML12 hepatocytes as described previously [10]. Cells were lysed after pretreatment with or without HK (10 μM) for 12 h. The lysates were centrifuged at 12,000 g for 10 min at 4 °C after incubating in ice for 10 min. The protein concentration was determined and adjusted to 3 μg/μL using RIPA lysis buffer. Cell lysates (50 μL) were transferred to new tubes and heated for 3 min at various temperature (50-90°C) on a thermal cycler. After standing in ice for 10 min, soluble proteins were obtained by centrifugation at 12,000 g for 20 min at 4 °C and analyzed by western blotting [24].
Mitochondrial membrane potential assay The uorescent dye Rhodamine123 was employed to detect the mitochondrial membrane potential. Speci cally, AML12 cells were cultured in the presence or absence of HK, and stained with Rhodamine123 (10 μM) for 10 min. Then, cells were washed twice with PBS, trypsinized and collected into a 1.5 mL tube. The change of membrane potential was qualitatively observed on an In Cell Analyzer 2000 (GE Healthcare Life Sciences, Chicago, IL, USA).

Intracellular reactive oxygen species (ROS) detection
Intracellular ROS levels were detected using DCFH-DA as previously described [25]. Brie y, cells (1 × 10 5 ) were seeded into 96-well black multitier plate (clear bottom) and then cultured overnight. The cells were treated with or without HK. After 12 h, the cells were incubated with DCFH-DA (10 μM) at 37 °C in the dark for 15 min. Fluorescence intensity was analyzed through FACSCalibur ow cytometry (BD, Lake Franklin, NJ, USA).

Isoproterenol-stimulated lipolysis
The lipolysis activity of AML12 cells was measured as described previously [26]. Cells were incubated with 10 μM isoproterenol (stimulated condition) or DMSO (basal condition) for 2 h. Subsequently, the medium was collected and heated at 85 °C for 10 min. Clear supernatant (10 μL), acquired through centrifugation, was used to determine the free glycerol content using Free Glycerol Reagent. Lipolysis activity was represented by glycerol concentrations and normalized by protein concentration.
Immunoprecipitation Immunoprecipitation was performed as described previously [27]. Brie y, cell lysates (3 mg protein) were mixed with the indicated antibody (2 μg) at 4 °C overnight. Then protein A/G-agarose beads (20 μL) were added to the mixture, which was incubated on a rotator for 4 h at 4 °C. Immune complexes were washed twice with lysis buffer supplemented with complete mini-protease inhibitor cocktail after washing the beads for three times with PBS. Bound proteins were boiled in sample preparation buffer for 5 min and then immunoblotting was conducted.

Statistical analysis
All experimental data were expressed as mean ± S.D., and sample size for each experiment corresponds to three biological replicates. Data analysis was nished on GraphPad Prism-6 (GraphPad Software, San Diego, CA, USA), where signi cant differences between groups were evaluated by one-way analysis of variance (ANOVA) followed by Dunnet's multiple comparisons test (p < 0.05 was considered as signi cant differences). Where statistical signi cance is evaluated, variance between groups is con rmed to be similar between comparison groups (control vs. experimental) and the statistical analysis is considered appropriate.

Results
Honokiol attenuates lipid accumulation in lipotoxic hepatocytes through promoting autophagy P/O (palmitic acid and oleic acid mixture) is widely used to induce lipotoxicity in vitro baecause of its same effect in inducing steatosis and lower cytotoxicity than palmitic acid alone [28,29]. Herein, lipid accumulation in AML12 cells was realized by inducement with P/O (the ratio of oleic acid to palmitic acid is 1:2). Firstly, we evaluated the effects of HK against P/O-induced lipid accumulation in AML12 cells. HK did not exhibit obvious cytotoxicity on AML12 cells up to 10 µM [21]. Interestingly, a 2.7-fold increase of lipid content was observed after P/O stimulation, and HK attenuated this effect dose-dependently, which was comparable with the positive control resveratrol, as indicated by Nile red staining and its quantitative analyses (Fig. 1A, 1B). P/O-induced increase of TGs in AML12 cells was also reversed by HK treatment (Fig. 1C).
Impaired autophagy results in increased lipid storage in hepatocytes [30]. As shown in Fig. 1D, after P/Otreatment, there is a shrinkage in Beclin1 level and the ratio of LC3-II/LC3-I to approximately 48-75%, whereas p62 level was elevated to 267%, compared with those of the control cells, suggesting impaired autophagy in AML12 cells. Intriguingly, HK treatment reversed the above changes in dose-dependent manners (Fig. 1D). Meanwhile, HK also enhanced autophagy in unstimulated AML12 cells (Fig. 1D). To see whether HK enhanced autophagic ux, we infected the AML12 cells with mRFP-GFP-LC3 adenovirus to label autophagosomal formation. As shown in Fig. 1E and 1F, impaired autophagy was re ected by the decreased both red and green puncta after P/O treatment. More mRFP-LC3 puncta were observed in HKtreated cells as expected, suggesting that autophagic ux was improved with undisturbed lysosomal function and/or autophagosome-lysosome fusion. Furthermore, the uorescent images indicated that autophagosome formation, which was largely co-localized with LDs (Fig. 1G), was anhanced with HK. These results indicated that HK mitigates lipid accumulation in lipotoxic hepatocytes through promoting autophagy.
Honokiol attenuates lipid accumulation through SIRT3-mediated autophagy SIRT3 overexpression protects hepatocytes from lipotoxicity though promoting lipophagy and chaperonmediated autophagy to [10]. Interestingly, HK treatment dose-dependently increased SIRT3 level in P/Otreated AML12 cells ( Fig. 2A). To experimentally verify the interaction between HK and SIRT3 deacetylase, CETSA was performed on AML12 cells treated with or without HK. Compared to the control cells, the thermal stability of SIRT3 was strongly enhanced by HK at various temperatures (Fig. 2B). To verify the interaction pattern between HK and SIRT3, docking analysis was conducted. Clustering analysis showed the predominant cluster had the lowest binding energy with the best pose owing a − 7.2 kcal/mol (Fig.   2C). HK had hydrophobic interaction with amino acid residues (PHE294, GLU323 and VAL324) and NAD + (Fig. 2D). Additionally, it was hydrogen bonded with an oxygen on the NAD + (Fig. 2D).
To evaluate the role of HK-driven SIRT3 in reducing lipid accumulation in hepatocytes, thr SIRT3 knocking down AML12 cell line (SIRT3KD) was generated using shRNA targeting SIRT3. As expected, SIRT3 silencing partially blocked the lipid lowering effects of HK in P/O-stimulated AML12 cells (Fig. 2E). HK treatment increased the expression of LC3II, and reduced lipid content and LD size in P/O-treated AML12 cells; whereas, silencing of SIRT3 almost abrogated the effects of HK (Fig. 2F).
Next, we determined whether HK treatment is su cient for inducing lipophagy and the pharmacological activation of SIRT3 plays a role in this process. The LD fraction was isolated from scrambled and SIRT3KD AML12 cells treated with or without HK, and enrichment of LC3-II, Beclin1 and decreased p62 were observed in LDs, but not in homogenates after treatment of HK; and deletion of SIRT3 almost reversed HK-driven activation of autophagy in isolated LDs (Fig. 2G). These observations indicated HK treatment induced lipophagy rather than bulky autophagy to alleviate lipid accumulation and SIRT3 is required in HK-induced lipophagy.
Honokiol alleviates lipid accumulation through SIRT3-AMPK-induced autophagy SIRT3 activates autophagy through the AMP-activated kinase (AMPK) pathway in palmitate-stressed hepatocytes [10]. To elucidate the mechanism of HK on SIRT3-mediated autophagy, the phosphorylation level of AMPK was determined in HK treated hepatocytes. The HK dose-dependently increased the phosphorylated AMPK in P/O treated AML12 cells as expected (Fig. 3A). Compound C (CC), an inhibitor to AMPK, remarkably diminished the effect of HK on activating autophagy (Fig. 3B). As shown in Fig. 3C and 3D, treatment of CC reversed the reducing effect of HK on lipid and TG content, supporting that HK alleviated lipid accumulation in AML12 cells via activating AMPK signaling pathway. These results suggested that the effect of HK on lowering lipid accumulation was mediated through SIRT3-AMPKmediated autophagy.

Honokiol attenuates lipid accumulation by restoring mitochondrial function
Next, we assumed the lipid lowering effect of HK was involved in enhanced mitochondrial function. Therefore, we determined the mitochondrial membrane potential by using Rhodamine 123 staining. The results showed P/O stimulation disrupted mitochondrial membrane potential, whereas HK-treated cells exhibited higher mitochondrial membrane potential, suggesting improved mitochondrial function (Fig.  4A). Meanwhile, lipid challenge lead to high level of intracellular ROS and reduced mitochondrial. HK treatment alleviated oxidative stress and slightly enhanced mitochondrial biogenesis in P/O-treated AML12 cells (Fig. 4B and 4C). Activation of autophagy not only shifts lipids to the lysosome for degradation by acid lipases, but enhances lipolysis by neutral lipases [10]. HK enhanced lipolysis in a dose-dependent manner in isoproterenol-stimulated cells, but not the control cells (Fig. 4D). Long-chain acyl-CoA dehydrogenase (LCAD) is a deacetylating substrate of SIRT3 [31]. HK decreased the acetylated LCAD level in a dose-dependently, but did not change the total LCAD level (Fig. 4E). Taken together, HK rescues hepatocytes from P/O-induced lipid accumulation by maintaining mitochondrial function and promoting lipolysis.

Discussion
Increasing evidence has uncovered a positive connection between lipophagy and the onset of NAFLD. Organisms regulate FFAs release to supply metabolic demand by lipophagy [32]. Impaired autophagy leads to excessive lipid accumulation in liver to cause hepatic steatosis [33,34]. HK has been found to activate autophagy in several cancer cells [35][36][37]. Herein, we found HK attenuates lipid accumulation in lipotoxic hepatocytes through promoting SIRT3-AMPK-induced autophagy and mitochondrial function. To fully verify the role of HK in treating NAFLD, diet-induced fatty liver mice model and/or genetically obese mice model should be recruited in the future.
Autophagy accounts for a major part of lipolysis in liver. Consequently, autophagy blockage through knockdown of the key autophagic genes like Atgs in hepatocytes, led to an increase of LDs in cells even under normal nutritional conditions [38]. Interestingly, impaired autophagy further deteriorated LDs accumulation, leading to hepatotoxicity and severe steatosis [39]. In our current study, HK was found to activate lipophagy, stimulate lipolysis under isoproterenol treated condition and decrease the acetylated LCAD level. These results indicated that HK protects hepatocytes against lipotoxicity through enhancing lipophagy and lipolysis.
Liver contains a large number of mitochondria, which are the predominant source of intracellular ROS.
Excessive ROS accumulation results in cell death through the oxidation of polyunsaturated fatty acids [40]. Mitochondrial homeostasis was interrupted when the hepatocytes were exposed to P/O stimulation, accompanied with decreased mitochondrial content and increased ROS production. Interestingly, high dose of HK maintained mitochondrial integrity in hepatocytes under lipotoxic stress. In fact, mitochondrial biogenesis acts as a critical factor for mitochondrial quantity, and HK was found to facilitate the process by targeting PGC-1α [41]. Herein, HK did not affect mitochondrial biogenesis, but enhanced mitochondrial function in lipotoxic hepatocytes. SIRT3 may also be involved in mitochondrial renewal and hepatocyte proliferation through mitophagy mechanisms [42]. Future studies are required to fully elucidate how HK regulates mitophagy in the context of hepatocellular lipotoxicity.
AMPK directly stimulates mitochondrial energy production and strengthens mitochondrial biogenesis [43]. The current data showed that HK promoted the phosphorylation of AMPK, which could directly activate autophagy. Inhibition of AMPK reversed the lipid lowering effect of HK. In general, HK activates autophagy and attenuates lipid accumulation in P/O-treated hepatocytes through activating AMPK.
The most widespread and prevailing model describing the development of NAFLD is the "multiple-hit hypothesis", where the " rst hit", hepatic lipid accumulation, induces lipotoxicity or steatosis, rendering liver more vulnerable to "subsequent hits" injury, such as mitochondrial dysfunction and oxidative stress, which in turn leads to steatohepatitis and cirrhosis, and eventually hepatocellular carcinoma [44]. In the previous study, we reported that HK scavenges excessive ROS and repairs cellular damages in oxidative injured hepatocytes. Herein, we further found that HK activates lipophagy to promote LD breakdown, leading to reduced lipotoxicity in hepatocytes. Taken together, HK might protect hepatocytes against oxidative stress and alleviate lipotoxicity, rendering it a potential candidate to treat NAFLD against multiple hits.

Conclusions
In conclusion, we veri ed HK protects hepatocytes against lipotoxic stress through enhancing SIRT3-AMPK-induced lipophagy, and maintain mitochondrial morphology and integrity (Fig. 5). HK could be a potential candidate in the treatment of NAFLD.