Catalpol induces autophagy and attenuates liver steatosis in ob/ob and high-fat diet-induced obese mice

CAT attenuated liver steatosis in ob/ob and HFD mice

To explore the effect of CAT on fatty livers, we administrated CAT or saline (control) to ob/ob and HFD mice intragastrically and analyzed liver/body weight ratios, liver tissues, and serum samples. In ob/ob mice, body weights were unchanged after CAT administration, but liver weight, liver/body weight ratios, serum TG and TC levels, and hepatic TG and TC content were all reduced (P <0.05) (Figure 1A–1C). H&E and Oil Red O staining indicated that hepatosteatosis also improved after CAT administration in ob/ob mice (Figure 1D). These data suggested that CAT alleviated liver steatosis, serum and liver lipid phenotype in obesity. Interestingly, CAT treatment downregulated expression of several hepatic lipogenic genes, such as ACC1α and FAS (P <0.05), and upregulated fatty acid oxidation genes, such as PPARα and CPT1 (P <0.05) (Figure 1E).

Figure 1. CAT ameliorated hepatic steatosis in ob/ob mice. Eight week-old ob/ob mice were treated with CAT (50 mg/kg/d) or vehicle by oral gavage for 4 weeks. (A) Gross images of liver tissue and changes in liver and body weights. (B) Serum TG and TC levels. (C) Liver TG and TC content normalized to total protein. (D) Representative photomicrographs of liver sections stained with H&E and Oil Red O. Scale bars: 50 μm. (E) mRNA expression levels of hepatic lipogenic genes ACC1α and FAS and fatty acid oxidation genes PPARα and CPT1. Data are expressed as fold-change relative to vehicle-treated ob/ob mice. (F) Representative western blot analysis of LC3-II, Sqstm1/P62, and Becn1 proteins. Unpaired two-sided t-tests were used for statistical comparisons to controls. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. vehicle-treated ob/ob mice.

In addition, after CAT administration, protein levels of LC3-II, a well-established marker of autophagy induction, increased, while levels of SQSTM1/p62 protein, which accumulates when autophagy is suppressed, decreased (Figure 1F). These results indicate that CAT might prevent liver steatosis by inducing autophagy.

Next, we examined whether CAT also had beneficial effects on liver steatosis in a HFD-induced obesity mice model. Remarkably, liver weights, liver/body weight ratios, serum TG, TC, ALS, and ALT levels, and liver TG and TC content all decreased after CAT administration in HFD mice (P <0.05) (Figure 2A–2E). Furthermore, as was the case in ob/ob mice, ACC1α and FAS mRNA expression decreased, while PPARα and CPT1 expression increased, after CAT administration in HFD mice (Figure 2F). LC3II protein levels also increased, while SQSTM1/p62 protein levels decreased, after CAT administration in HFD mice (Figure 2G, 2H). Because free fatty acid (FFA)-induced lipotoxicity and resulting cell death are important features of the pathogenesis of NAFLD [18], we next investigated the effect of CAT on caspase-3 (CASP3) activity in HFD mice. Indeed, HFD was cytotoxic to hepatocytes, and CAT significantly attenuated this cytotoxicity (Supplementary Figure 1). Together, these results suggest that CAT ameliorates HFD-induced hepatic steatosis by activating autophagy.

Figure 2. CAT ameliorates liver steatosis in HFD-fed mice. Mice were treated with CAT (50 mg/kg/d) or vehicle by oral gavage for 4 weeks. (A) Gross images of liver tissue and changes in liver and body weights. (B) Serum TG and TC levels. (C) Serum ALT and AST levels. (D) Liver TG and TC content normalized to total protein. (E) Representative photomicrographs of liver sections stained with H&E and Oil Red O. Scale bars: 50 μm. (F) mRNA expression levels of hepatic lipogenic genes ACC1α and FAS and fatty acid oxidation genes PPARα and CPT1. Data are expressed as fold-change relative to NCD mice. (G) Representative western blot analysis of LC3B-II and Sqstm1/P62. (H) LC3B-II and P62 band densities were normalized to tubulin. Means ± SD were calculated from three independent experiments. One-way ANOVAs with Tukey post-hoc tests were performed. n =5 per group. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

CAT induced autophagy in hepatocytes

Previous reports have suggested that autophagy plays a crucial role in hepatic steatosis and that it is suppressed in the livers of NASH patients and HFD mice. To evaluate the effects of CAT on autophagy, hepatic steatosis, and the underlying mechanisms, HepG2 cells were exposed to different concentrations (0.1, 1, or 10 μg/mL) of CAT for 24 h or treated with 10 μg/mL CAT for 6, 12, or 24 h. CAT treatment increased LC3-II levels and decreased SQSTM1/p62 levels in a dose- and time- dependent manner (Figure 3A, 3B). We next evaluated the effect of CAT on autophagy using fluorescence microscopy in GFP-LC3 transduced hepatocytes. LC3-II levels markedly increased in response to 24 h of CAT administration (Figure 3C). Additionally, electron microscopy revealed that more autophagic vacuoles formed in hepatocytes treated with CAT compared to controls (Figure 3D). To investigate the effects of CAT on autophagy flux, HepG2 cells were treated with CAT for 24 h with or without CQ (50 μM). Following exposure to CAT, LC3B-II levels increased, and SQSTM1/p62 levels decreased, in hepatocytes. CQ alone significantly increased LC3-II and SQSTM1/p62 levels, while coadministration with CAT further increased LC3-II levels and decreased SQSTM1/p62 accumulation (Figure 3E). Consistent with that result, GFP-LC3-II expression levels decreased, while free-GFP expression levels increased, in hepatocytes treated with CAT (Supplementary Figure 2). We also observed that CAT significantly upregulated autophagy-related genes (including Atg7, Atg5, Becn1, Ulk1, and Lamp1) and TFEB in HepG2 cells (Figure 3G). Together, these data indicate that CAT enhanced autophagy in hepatocytes.

Figure 3. CAT induced autophagy in hepatocytes. (A) Dose-dependent induction of autophagy by CAT. Representative immunoblot analysis of LC3-II and SQSTM1 expression in lysates from HepG2 cells that were treated with CAT or control solvent (dimethyl sulfoxide, DMSO) at the indicated concentrations for 24 h. (B) Time-dependent induction of autophagy by CAT. Immunoblot detection of LC3-II and SQSTM1 expression in HepG2 cells treated with CAT (10 μg/mL) or DMSO for the indicated time. (C) HepG2 cells were transduced with an GFP-LC3 plasmid for 48 h and then treated with CAT (10 μg/mL) for 24h. Cells were observed by fluorescence microscopy to evaluate the number expressing GFP-LC3. (D) Representative electron microscopic pictures of HepG2 cells treated with CAT for 24 h. Arrows indicate autophagosomes. (E) Immunoblot analysis of LC3-II and SQSTM1 expression in HepG2 cells treated with 10 μg/mL CAT for 24 h in the absence or presence of 50 mM chloroquine (CQ) for the last 2 h. (F) qPCR analysis of autophagy-related and lysosomal genes in CAT-treated HepG2 cells. Means ± SD were calculated from three independent experiments. ^*P < 0.05, ^**P < 0.01.

CAT induced autophagy via the AMPK-TFEB pathway

Previous studies report that AMPK activates autophagy and reduces lipid accumulation invivo and invitro [19, 20]; we therefore evaluated the effects of CAT on AMPK activation in HFD-fed mice (Figure 4A). Notably, CAT induced AMPK phosphorylation and activated autophagy in HepG2 cells (Figure 4B). In addition, AMPK phosphorylation, but not levels of AMPK itself, decreased significantly in the presence of PA and increased after CAT treatment (Figure 4C). TFEB activates the transcription of genes that promote lysosomal biogenesis and regulates autophagy upon translocation to the nucleus. We therefore examined whether CAT treatment activated autophagy by promoting translocation of TFEB. Western blot analysis showed that CAT increased nuclear TFEB translocalization in hepatocytes (Figure 4A, 4B. Nuclear TFEB levels in hepatocytes after CAT administration as determined by fluorescence analyses were consistent with these results (Figure 4D). We then tested whether AMPK was required for CAT-induced autophagy and nuclear TFEB translocation. Inhibition of AMPK by CC reduced autophagic activity as evidenced by decreased LC3-II levels and increased SQSTM1/p62 levels (Figure 4E) and by a significant decrease in the number of GFP-LC3-positive vesicles (Figure 4F). Furthermore, fluorescence analyses and Western blots indicated that CC abrogated CAT-induced nuclear translocation of TFEB (Figure 4G, 4H). Collectively, these findings indicate that CAT induces autophagy via an AMPK/TFEB-related pathway.

Figure 4. CAT induced autophagy via the AMPK-TFEB pathway. (A) Immunoblot detection of TFEB, AMPK, and AMPK phosphorylation levels in livers of mice. (B) Immunoblot detection of LC3-II, SQSTM1, TFEB, AMPK, and AMPK phosphorylation levels in HepG2 cells treated with CAT (10 μg/mL) for 24 h. (C) Immunoblot analysis of AMPK and p-AMPK levels in HepG2 cells treated with 0.3 mM palmitate (PA) and 10 μg/mL CAT for 24 h. (D) Fluorescence microscopy images of nuclear TFEB in HepG2 cells treated with 10 μg/mL CAT for 24 h. Scale bars: 25 μm. (E) Immunoblots of LC3-II, SQSTM1, AMPK, and AMPK phosphorylation in HepG2 cells treated with CAT (10 μg/mL) and Compound C (CC, 10 μM) for 24 h. (F) Numbers of GFP-LC3 in HepG2 cells expressing GFP-LC3 after treatment with or without CAT (10 μg/mL) and CC (10 μM) for 24 h were evaluated using fluorescence microscopy. Scale bars: 25 μm. (G, H) Fluorescence microscopy images and Immunoblot analysis of nuclear TFEB in HepG2 cells treated with 10 μg/mL CAT and Compound C (CC, 10 μM) for 24 h. Scale bars: 25 μm. ^***P < 0.001.

CAT ameliorated liver steatosis via AMPK-induced autophagy

To determine whether the protective effects of CAT against liver steatosis were autophagy-dependent, an invitro model of NAFLD was established by treating HepG2 cells with PA for 24 h (Supplementary Figures 3A, 4B). Flow cytometry indicated that apoptosis levels increased significantly in cells treated with PA (Supplementary Figure 3C). Lipid staining and liver lipid content analyses showed that CAT significantly attenuated PA-induced lipid accumulation in hepatocytes; this protective effect of CAT decreased in the presence of CQ (Figure 5A, 5B). We then examined whether the inhibitory effects of CAT on lipotoxicity-mediated apoptosis was dependent on autophagy in hepatocytes. CAT treatment attenuated lipoapoptosis, as evidenced by decreased cleaved caspase-3 and PARP expression levels, but these effects were eliminated by co-treatment with CQ (Supplementary Figure 4A). These results were confirmed by flow cytometry analysis (Supplementary Figure 4B). Together, these data indicate that CAT inhibits lipid accumulation and lipoapoptosis by promoting autophagic flux.

Figure 5. CAT ameliorated liver steatosis by inducing autophagy via AMPK. Cells were treated with 0.3 mM PA and 10 μg/mL CAT for 24 h in the presence or absence of 50 mM chloroquine (CQ) for 2 h. HepG2 cells were stained with Oil Red O (A), and intracellular TG and TC were quantitatively analyzed (B). Scale bars: 25 μm. HepG2 cells treated with 0.3 mM PA, 10 μg/mL CAT for 24 h, and 10 μM compound C (CC) for 24 h. Cells were stained with Oil Red O (C), and intracellular TG and TC were quantitatively analyzed (D). Scale bars: 25 μm. (E) Expression of hepatic lipogenic genes ACC1α and FAS and fatty acid oxidation genes PPARα and CPT1 was quantitatively analyzed (n=3). (F) A schematic illustration demonstrating the role of CAT in NAFLD and the effect of CAT on autophagy. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Next, we investigated whether the AMPK-dependent pathway identified above was involved in CAT-dependent reversal of liver steatosis. CAT markedly reduced PA-induced intracellular lipid accumulation, and co-treatment with CC attenuated this effect, indicating that the protective effects of CAT against hepatic steatosis are mediated by the AMPK pathway (Figure 5C, 5D). More importantly, incubation of hepatocytes with CC completely abrogated CAT-induced upregulation of the fatty acid metabolism genes FAS, ACC1, CPT1, and PPARα (P <0.05) (Figure 5E).

Reagents

The following chemicals were used: Dulbecco's Modified Eagle Medium (DMEM, Gibco), fetal bovine serum (FBS, Gibco), pre-stained protein markers from Fermentas (Thermo Fisher Scientific, 26616), palmitate (PA, Sigma-Aldrich, P9767), chloroquine (CQ; Sigma-Aldrich, C6628), Oil Red O (Sigma-Aldrich, O1391), CAT (98% purity, MedChem Express, HY-N0820), Compound C (CC, MedChem Express, HY-13418A).

Mouse models and CAT treatment

Eight-week old male ob/ob mice were purchased from Beijing Huafukang Bioscience Co. Inc. (Beijing, China). Animals were maintained under a 12 h light-dark cycle with free access to food and water. The mice were acclimatized to laboratory conditions for 1 week before the study and then divided into two groups: the control group (n=5) received a daily oral gavage of the vehicle (100 μL of sterile saline), while the CAT group (n=5) received CAT at 100 mg/kg of body weight. Mice were sacrificed after 4 weeks of treatment, blood was collected via the tail vein, and liver tissues were immediately removed, weighted, and stored at -80°C for further analysis.

Seven-week old male C57BL/6 mice obtained from Beijing Huafukang Bioscience Co. Inc. (Beijing, China) were housed in plastic cages under same conditions described above. After a 1 week acclimatization period, they were divided into three groups: one group received normal chow diet (NCD), and the two other received a HFD (60 Kcal% fat, D12492, Research diet, New Brunswick, NJ, USA) for 12 weeks with or without 50 mg/kg/day CAT dissolved in saline via gastric gavage for the last 4 weeks (n=5 in each group). After starvation for 6 h, mice were anesthetized with pentobarbital (40 mg/kg body weight) and blood and livers were collected, processed, and stored at -80°C. All animal experiments were approved by the Institutional Animal Research Committee of Tongji Medical College, Huazhong University of Science and Technology.

Cell culture and treatment

HepG2 cells were cultured with DMEM supplemented with 10% FBS at 37°C in 5% CO₂. To establish a cellular model of NAFLD, HepG2 cells were treated with PA at a concentration of 0.3 mM for 24 h [33]. For the CAT treatment experiment, hepatocytes were exposed to CAT at a concentration of 10 μg/mL for 24 h. CQ (50 μM) was added for the last 2 h [23]. To study the effect of AMPK inhibition on translocation and phosphorylation of TFEB or autophagy, cells were treated with 10 μM CC for 24 h [20].

Quantitative PCR analysis

Total RNA was isolated from HepG2 cells and liver tissues using Trizol reagent (Invitrogen, 15596018) according to the manufacturer’s protocol. Two micrograms of total RNA were reverse transcribed using a FastQuant RT kit (Tiangenbiotech, Beijing, China, KR106). qPCR analysis was performed using Fast SYBR^® Green Master Mix (Thermo Fisher Scientific (Waltham, MA, USA) on a StepOne Plus™ Real-Time PCR System (Applied Biosystems). The sequences of primers used are listed in Supplementary Table 1. Relative gene expression levels were normalized to GAPDH expression level and calculated using the 2^−ΔΔC method.

Western blot analysis

Proteins were extracted from HepG2 cells and liver tissues. About 20-50 μg of protein were separated on 12% sodium dodecyl sulfate–polyacrylamide gels (SDS-PAGE), transferred to PVDF membranes, and then blocked with 5% skim milk solution in Tris buffered saline containing 0.1% Tween-20 for 1.5 h at room temperature. Western blotting was performed using the following primary antibodies: anti-sequestosome 1 (SQSTM1/P62, Santa Cruz Biotechnology, sc-28359), anti-LC3 (Sigma-Aldrich, L7543), anti-Tubulin (Santa Cruz Biotechnology, sc-73242), anti-TFEB (Abcam, ab220695), anti-AMPK (Santa Cruz Biotechnology, sc-74461), anti-Phospho-AMPKα (Thr172, Cell Signaling Technology, #50081), anti-Caspase-3 (Cell Signaling Technology, #9665), anti-PARP (Santa Cruz Biotechnology, sc-365315). Blots were detected using a chemiluminescence system (Bio-Ras Laboratories, Berkeley, CA, USA). Band intensities were densitometrically analyzed using ImageJ 1.8.0 Software.

Transfection and immunofluorescence

HepG2 cells were transfected with GFP-LC3 plasmid kindly provided by Fuqing Hu (Huazhong University of Science and Technology, Wuhan, China) for 48 h using Lipofectamine 2000 (Invitrogen/Life Technologies). The cells were then incubated with either CAT (10 μg/mL) or CC (10 μM) for 24 h. To identify nuclear translocation of TFEB, HepG2 cells were exposed to CAT (10 μg/mL) for 24 h. For immunofluorescence, cells were fixed in 4% paraformaldehyde, permeabilized using 0.1% Triton X-100, and blocked by incubating in PBST with 10% goat serum and 3% BSA for 1 h at room temperature. Samples were then incubated with primary antibody overnight at 4°C followed by incubation with secondary antibodies for 3 h. Images were captured with a confocal microscope (LSM700; Carl Zeiss Inc., Oberkochen, Germany).

Transmission electron microscopy

Liver samples or cells were fixed with 2% glutaraldehyde/paraformaldehyde overnight at 4°C, treated with 1% osmiumtetroxideina for 1 h at room temperature, and dehydrated in graded ethanol solutions according standard procedures. Samples were then embedded in Epon 812 (Electron Microscopy Sciences,100503–876) and sectioned using a Reichert Ultracut E microtome (Leica Microsystems, Buffalo Grove, IL, USA). EM images were acquired using a H7600 transmission electron microscope (Hitachi, Tokyo, Japan).

Hepatic and cellular TG and TC measurement

Intracellular and intrahepatic TG and TC content was measured using commercial kits (Jiancheng Technologies Inc, Nanjing, China) according to the manufacturer’s recommended protocols.

Histopathological examination

Liver sections were fixed in 4% paraformaldehyde, embedded in paraffin, and then stained using hematoxylin and eosin (H&E) for histological examination. To determine lipid droplet accumulation, frozen liver sections (8 μm) were incubated with freshly diluted Oil Red O staining solution for 10 min, washed, and then counterstained with hematoxylin for 5 min. Cells were fixed in 4% paraformaldehyde for 15 min, stained with Oil Red O staining solution for 10 min, washed with 60% isopropanol, and stained with hematoxylin for 5 min. The histological features of the samples were observed and imaged at 400 × magnification using light microscopy (Olympus, Japan).

Biochemistry assay

Serum TC, TG, aspartate transaminase (AST), and alanine transaminase (ALT) were measured using a colorimetric diagnostic kit (Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions.

Caspase 3 activity assay

Cell viability was assessed using a caspase 3 activity assay kit (Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer’s protocol. Liver tissues (5 mg) were homogenized in 100 μL lysis buffer. Next, 50 μL lysates were exposed to 10 μL of the caspase 3 substrate in assay buffer (total volume: 100 μL) for 120 min at 37°C. Optical density was measured using an ELISA reader at an absorbance of 405 nm.

Flow cytometry

To measure lipoapoptosis, cells were cultured with either PA (0.3 μM) or CAT (10 μg/mL) for 24 h. CQ (50 μM) was added for the last 2 h, followed by staining with Annexin V and 7-amino-actinomycin D (7-AAD; BD Biosciences, Mississauga, ON); flow cytometry was then performed using a FACSCanto II (BD Biosciences, Mississauga, ON). Early-stage apoptotic cells are PE Annexin V-positive and 7-AAD-negative, while late-stage apoptotic cells are both PE Annexin V- and 7-AAD-positive.

Statistical analysis

The data are expressed as mean ± SD. Unpaired two-sided t-tests were used for comparisons between two groups. One-way analysis of variance (ANOVA) with post hoc Bonferroni tests was used for comparisons between multiple groups. P values < 0.05 were considered statistically significant.