D-Mannose Regulates Hepatocyte Lipid Metabolism via PI3K/Akt/mTOR Signaling Pathway and Ameliorates Hepatic Steatosis in Alcoholic Liver Disease

D-mannose (purity ≥ 99%, Cat.#M2069) and ethanol (purity≥ 99.8%, 51976) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rapamycin (purity = 99.30%, S1039), LY294002 (purity = 99.84%, S1105), 740 Y-P (purity = 98.38%, S7865) and MHY1485 (purity = 99.09%, S7811) were purchased from Selleck Chemicals (Houston, Texas, USA). Liquid Standard Diet (TP4020C), Lieber-DeCarli Control Liquid Diet (TP4030C), and Lieber-DeCarli Ethanol Liquid Diet (TP4030D) were supplied by TROPHIC Animal Feed High-Tech Co. Ltd (Hai’an, Jiangsu, China). The antibodies against SREBP1c (AF-6283), PPARα (AF5301), ACC1 (AF6421), P110 of PI3K (AF-5112) were all from Affinity (Ancaster, ON, Canada). Anti-CPT1A (15184-1-AP), anti-ACOX1 (10957-1-AP), anti-FASN (10624-1-AP), anti-P85 (60225-1-Ig), anti-PPARγ (16643-1-AP), anti-PPARα (15540-1-AP) used in Figure 6D were obtained from Proteintech (Chicago, IL, USA). The antibodies against Akt (4691), phosphor-Akt (4060), mTOR (2983), phosphor-mTOR (5536) were from Cell Signaling Technology (Danvers, MA, USA).

C57BL/6 mice (male, 8-10 weeks old) were purchased from the Experimental Animal Center of Southern Medical University (Guangzhou, China). All mice were housed under a 12-h light/dark cycle in a specific pathogen-free animal condition with a controlled temperature (20-25°C) and humidity (50 ± 5%). All animal experiments in this study were approved by the Southern Medical University Experimental Animal Ethics Committee (No. L2020128).

The chronic-binge mouse model was established based on the methods of previous studies with minor modifications (29). Briefly, mice were fed a standard liquid diet for 3 days, then randomly divided into different groups as follows: Pair (Lieber-DeCarli control liquid diet); EtOH (Lieber-DeCarli ethanol liquid diet; ALD group); Pair+Man (Lieber-DeCarli control liquid diet supplemented with 3% (w/v) mannose); EtOH+Man [Lieber-DeCarli ethanol liquid diet supplemented with 1%, 2%, 3% (w/v) mannose (27)]. The mice in the EtOH and EtOH+Man groups were fed the Lieber-DeCarli liquid diet containing increasing 1% to 4% (w/v) ethanol for the first 6 days and then the diet with 5% ethanol for 10 days. On day 11, mice fed ethanol before were gavaged a single dose of ethanol (5 g/kg body weight, 31.5% ethanol), while mice fed control diet were gavaged isocaloric dextrin maltose. Subsequently, the mice were sacrificed nine hours post gavage. Blood samples were obtained from the eye socket. A portion of the liver tissues was fixed in 4% neutral buffered formalin solution, and the remaining liver sections were immediately stored at -80°C.

Isolated primary hepatocytes from WT C57BL/6 mice (male, 8-12 weeks old) were obtained using a classical two-step in situ collagenase perfusion method as described previously with slight modifications (30). Briefly, the perfused liver was immediately excised and placed in a sterile dish containing RPMI 1640 medium (Gibco, United States). Then the cell suspension was filtered through a 70-μm nylon filter (BD Biosciences) and washed thrice by centrifugation at 50 × g for 3 min at 4°C. Subsequently, the cells were resuspended in the growth medium containing William’s E medium (Thermo Fisher, Carlsbad, CA, USA) supplied with 10% fetal bovine serum (FBS, Gibco, United States), 10 ng/mL epidermal growth factor (EGF, GenScript, Nanjing, China), 2 nM L-glutamine (Macklin, Shanghai, China), 200 nM insulin (Macklin, Shanghai, China) and 100 nM dexamethasone (Macklin, Shanghai, China), and then seeded on type I collagen-coated dish. After incubation at 37°C for 4 h, PMHs were collected and washed twice, and the medium was replaced with the fresh growth medium.

Cultured PMHs were treated with 200 mM ethanol (EtOH) (31, 32) or cell growth medium only (Ctrl), in the presence of different concentrations (1 mM, 2.5 mM, 5 mM, 10 mM) of mannose (Man) (26) or not for 24 h. In some experiments, inhibitors (33) or agonists (34, 35) of PI3K or mTOR (Dimethyl sulfoxide (DMSO) as control) was added two hours before ethanol exposure or mannose treatment.

Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), total cholesterol (TC), high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C) levels, and hepatic triglyceride (TG), total cholesterol (TC) contents were all measured according to the instructions of commercial assay kits from the manufacturer (Jiancheng Biotech, Nanjing, China).

The paraffin-embedded liver tissue blocks (n = 3 for each group) were cut into 5 μm slices sections and stained with hematoxylin and eosin (H&E). The frozen liver tissues (n = 3 for each group) were cut into 8 μm thick sections and then stained with Oil Red O. For immunohistochemical staining, liver tissue sections were deparaffinized and placed in a citrate buffer (pH 6.0) at 100°C for 10 min to antigen repair and then exposed to 3% H₂O₂ for 15 min to block endogenous peroxidase activity. Subsequently, sections were blocked with 5% normal goat serum for another 1 h at room temperature followed by incubated with primary antibodies at 4°C overnight. Immuno-reactivity was detected using the corresponding HRP-conjugated secondary antibody and visualized using a diaminobenzidine kit (Beyotime Institute of Biotechnology, Shanghai, China).

The cellular content of neutral lipids was detected according to the manufacturer’s instructions using lipophilic fluorescence dye BODIPY 493/503 (Invitrogen, Carlsbad, CA, USA). Briefly, cells were seeded on the 12-well culture plates containing cell-climbing slices pre-coated with collagen and incubated overnight. Cells were washed with Phosphate Buffered Saline (PBS) and fixed with 4% paraformaldehyde for 20 min at room temperature. Subsequently, cells were stained with 1 μg/mL BODIPY 493/503 dye for 30 min at 37°C, the nuclei were counterstained with 1 μg/mL Hoechst (CST) for 10 min. Then the slices were mounted on microscope glass slides and imaged immediately with a laser scanning microscope system (Nikon Eclipse Ni, Tokyo, Japan).

The protein of PMHs was homogenized in RIPA buffer containing protease inhibitor (Beyotime Institute of Biotechnology, Shanghai, China). Subsequently, the protein concentrations were measured using a BCA protein assay kit (Beyotime Institute of Biotechnology). Equivalent amounts of protein were separated by SDS-PAGE and then transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membrane was blocked with 5% bovine albumin (BSA) in Tris-buffered saline containing 0.05% Tween 20 and then incubated with the specific primary antibodies, followed by HRP-conjugated secondary antibody incubation. And the target proteins were visualized with enhanced chemiluminescence (Thermo Fisher, Carlsbad, CA, USA). The intensity of the protein band was quantified using ImageJ software.

The total RNA was extracted using TRIzol reagent (TransGene Biotech, Beijing, China) and then transcribed into cDNA using TranScript All-in-One First-Strand cDNA Synthesis SuperMix (TransGene Biotech), as instructed by the manufacturer. Real-time PCR was performed with an Eppendorf Realplex PCR system using TransStart Tip Green qPCR SuperMix (TransGene Biotech). The mRNA expression was normalized to the expression of the housekeeping gene β-actin. All primer sequences presented in Table 1 were from PrimerBank (36) and synthesized by Huada Gene Technology Co., Ltd (Shenzhen, China).

All data were expressed as mean ± SEM. Statistical significance was determined by the unpaired two-tailed t-test using GraphPad Prism 8.0 software (San Diego, CA, USA). Differences were considered statistically significant at p < 0.05.

To address the role of mannose supplement in ALD, we investigated the degree of liver injury and hepatic steatosis in pair-fed mice (Pair) or a chronic-binge ethanol feeding mouse model of ALD (EtOH), along with drinking-water supplemented with different concentrations of mannose (Man). As shown in Figure 1A and Table S1, enzymatic assays demonstrated that mannose administration significantly reduced serum ALT and AST levels that elevated in chronic-binge ethanol-fed mice. The H&E staining of liver sections showed that ethanol-fed mice displayed extensive hepatic injuries and steatosis, which were markedly attenuated by mannose administration (Figure 1B). Furthermore, alcohol consumption substantially elevated TG, TC, and LDL-C levels but reduced HDL-C level in serum or liver tissue, which could be significantly inhibited by oral mannose (3%) supplement (Figures 1C–E and Table S1). Further Oil Red O staining of liver tissue sections revealed that mannose remarkably reduced ethanol-induced hepatic lipid deposits, and 3% mannose has the most significant effect (Figure 1F). Additionally, we performed immunofluorescence staining of liver tissue sections with BODIPY and found that mannose co-administration markedly reduced lipid deposits that present predominantly in hepatocytes upon ethanol administration (Figure 1G). Collectively, these results indicated a potential protective role of mannose against hepatic steatosis in ALD.

Building upon the above findings in the mouse ALD model, we explored the exact role of mannose on hepatocytes in vitro. Therefore, we utilize an in vitro model of ALD established using ethanol-treated PMHs (32, 37, 38). Since most of the studies about the mannose supplement, mannose was added concurrently with other drugs or stimuli (24, 25), we simultaneously treated the PMHs with ethanol and indicated concentrations of mannose. Consistent with the above results in vivo, we demonstrated that mannose treatment significantly reduced serum ALT and AST activities and intracellular TG and TC levels in PMHs, which notably increased upon ethanol stimulation (Figures 2A, B and Table S2). Moreover, this effect was dose-dependent but with an effective plateau or saturation at concentrations higher than 5 mM (Figures 2A, B and Table S2). Further cellular staining with BODIPY 493/503 lipophilic fluorescent dye showed that ethanol notably elevated cellular neutral lipid contents deposited within lipid droplets in cultured PMHs, which significantly reduced upon mannose treatment (Figure 2C). These findings indicated that mannose could attenuate ethanol-induced lipid accumulation in PMHs.

Considering the above results showing that mannose attenuates ethanol-induced hepatocyte lipid accumulation in ALD, we were intrigued to clarify the underlying mechanisms. Firstly, we examined the mRNA levels and protein expression of crucial fatty acid oxidation (FAO)-related genes associated with lipid metabolism. As shown in Figures 3A, B, we observed notably reduced protein and mRNA levels of PPARα, a key controller of FAO (39), and its downstream FAO-related genes (ACOX1, CPT1) in isolated PMHs from ethanol-fed mice than that from pair-fed controls. However, oral mannose supplement significantly increased PPARα, ACOX1 and CPT1 levels in freshly isolated PMHs from ethanol-fed mice (Figures 3A, B). Further immunohistochemistry analysis also showed marked elevation of PPARα expression in liver sections from ethanol-fed mice upon mannose administration, although its expression notably decreased during ethanol feeding (Figure 3C). Similar results showed that mannose significantly increased protein and mRNA levels of PPARα, ACOX1 and CPT1 in PMHs upon ethanol treatment in vitro (Figures 3D, E). We also evaluated the effect of mannose on PPARγ, a nuclear receptor superfamily of ligand-inducible transcription factors involved in fatty acid uptake (40, 41). While the protein and mRNA levels of PPARγ in PMHs elevated upon ethanol stimulation, they were comparable between the mannose treated or untreated PMHs in vitro (Figure 3D). These data suggest that mannose might attenuate ethanol-induced lipid accumulation in hepatocytes via regulating lipid metabolism by upregulating fatty acid β-oxidation.

Besides fatty acid oxidation, alcohol-induced hepatic lipid accumulation is also regulated by lipogenesis (42). Therefore, we next investigated whether mannose disturbs the alcohol-induced de novo lipogenesis in hepatocytes. As shown in Figures 4A, B, oral mannose supplementation could reverse the alcohol-induced elevation of protein and mRNA levels of crucial lipogenic enzyme SREBP1c and its downstream lipogenic genes (ACC1, FASN) in isolated PMHs. Further immunohistochemistry staining determined notably reduced SREBP1c expression in liver tissue sections from ethanol-fed mice upon mannose administration compared to that without mannose supplement (Figure 4C). Accordantly, we demonstrated that in vitro mannose treatment could eliminate the ethanol-induced increased protein and mRNA levels of SREBP1c, ACC1 and FASN in cultured PMHs (Figures 4D, E). Therefore, these data suggest that mannose might ameliorate ethanol-induced hepatocyte lipid accumulation in ALD by regulating lipid metabolism by inhibiting lipogenesis.

Given that PI3K/Akt/mTOR signaling pathway plays a crucial role in regulating the lipid metabolic process (12, 43), we subsequently investigated whether mannose affected PI3K/Akt/mTOR signaling pathway activation. Western blotting analysis demonstrated that mannose supplement significantly downregulated alcohol-induced elevation of PI3K expression (subunit p85, p110), as well as Akt and mTOR phosphorylation in isolated PMHs from ethanol-fed mice (Figure 5A). Additionally, we also observed that mannose notably downregulated the ethanol-induced increased levels of PI3K (subunit p85, p110), as well as Akt and mTOR phosphorylation in PMHs upon ethanol treatment in vitro (Figure 5B). Therefore, these data indicate that mannose suppresses the ethanol-induced PI3K/Akt/mTOR signaling pathway activation in ALD.

Our data above point to the potential involvement of the PI3K/Akt/mTOR signaling pathway in mannose-mediated alleviation of lipid accumulation driven by ethanol-mediated imbalanced lipid metabolism in hepatocytes. To test this, we pretreated PMHs with specific inhibitors or agonists of the PI3K/Akt/mTOR signaling pathway ahead of mannose with/without ethanol treatment. Western blotting analysis showed that pretreatment with the mTOR-specific inhibitor, rapamycin, did suppress mTOR phosphorylation, whereas its agonist MHY1485 could trigger mTOR phosphorylation (Figure 6A). However, these pretreatments could eliminate the differences of ethanol-induced hepatocyte lipid accumulation between mannose treated or untreated PMHs, as determined by comparable intracellular TG and TC levels, cellular neutral lipid contents, protein and mRNA levels of FAO-related genes (PPARα, CPT1, ACOX1) and lipogenic genes (SREBP-1, ACC1, FASN) in these cells (Figures 6B–G). Similar results were observed when using the PI3K inhibitor LY294002 or its agonist 740 Y-P instead of the inhibitor and agonist of mTOR (Figure 7). Overall, these results confirmed that PI3K/Akt/mTOR singling is responsible for the inhibitory effect of mannose on lipid accumulation in hepatocytes.

The original contributions presented in the study are included in the article/. Further inquiries can be directed to the corresponding authors. 1