Name: investigating the protective effects of platycodin d on non-alcoholic fatty liver disease in a palmitic acid-induced in vitro model
Uploaded: 2022-12-02T15:00:00.000+00:00
Duration: 8 min 20 s
Description: Watch this Scientific Journal Video about Investigating the Protective Effects of Platycodin D on Non-Alcoholic Fatty Liver Disease in a Palmitic Acid-Induced In Vitro Model at JoVE.com

This work is supported by grants from the Chongqing Science and Technology Commission (cstc2020jxjl-jbky10002, jbky20200026, cstc2021jscx-dxwtBX0013, and jbky20210029) and the China Postdoctoral Science Foundation (No. 2021MD703919).

AML-12 cells (a normal mouse hepatocyte cell line) are used for the cell-based studies. The cells are obtained from a commercial source (see Table of Materials).
1. Pretreatment of the AML-12 cells to model NAFLD in vitro
<ol>
	<li>Maintain the cells in normal cell culture media (DMEM plus Ham&#39;s F12 [1:1] containing 0.005 mg/mL insulin, 5 ng/mL selenium, 0.005 mg/mL transferrin, 40 ng/mL dexamethasone, and 10% fetal bovine serum [FBS], see Table of Materials) at 37 &#176;C in a humidified atmosphere with 5% CO2.</li>
	<li>Seed the cells in 12-well plates (1 mL/well) with a density of 2.8 x 105 cells/well. 
	NOTE: All the cell digestion and medium exchange operations are performed in a biosafety cabinet to avoid contaminating the cells.</li>
	<li>Remove the culture medium of the cells after the overnight incubation (~10 h), and then wash the cells two times with 1 mL of serum-free media (per well).</li>
	<li>Divide the 12-well cell plate into four different treatment groups from left to right, including (1) the control group, (2) the PD-treated group, (3) the PA-treated group, and (4) the PA + PD-treated group. 
	NOTE: Three replicates of each experimental treatment group are arranged from top to bottom on the same 12-well cell plate.</li>
	<li>Add the normal cell culture media (1 mL/well) to the control group and the PD-treated group, and add the normal cell culture media (1 mL/well) supplemented with 300 &#181;M of PA to the PA-treated and PA + PD-treated group.</li>
	<li>Remove the culture medium of the cells after 24 h of incubation, and then wash the cells with 1 mL of serum-free media (per well) two times.</li>
	<li>Add normal cell culture media (1 mL/well) supplemented with a vehicle (dimethyl sulfoxide, DMSO, 0.1% v/v) to the control group; add normal cell culture media (1 mL/well) supplemented with 1 &#181;M PD to the PD-treated group; add normal cell culture media (1 mL/well) supplemented with 300 &#181;M PA to the PA-treated group; add normal cell culture media (1 mL/well) supplemented with 300 &#181;M PA and 1 &#181;M PD to the PA + PD-treated group.</li>
	<li>After incubation for an additional 24 h, investigate the protective effects of PD on cells using 2&#8242;,7&#8242;-dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining (step 2), JC-1 staining (step 3), and western blotting (step 4). 
	&#8203;NOTE: All the incubation operations are done at 37 &#176;C in a humidified atmosphere with 5% CO2.</li>
</ol>
2. Measurement of the change in ROS production
NOTE: The intracellular ROS levels in the cells are assessed based on the DCFH-DA staining assay.
<ol>
	<li>At the end of the treatment period (step 1.8), wash the cells with 1 mL of phosphate-buffered saline per well (1x PBS, pH 7.4) three times, and then stain the cells with 100 &#181;L of 10 &#181;M DCFH-DA per well (see Table of Materials). Incubate the cells in the dark for 30 min. 
	NOTE: If no obvious green fluorescence is observed after 30 min of incubation, the incubation time of the probe and cells can be appropriately increased (30-60 min). If overexposure of the green fluorescence value is observed after 30 min of incubation, the incubation time of the probe and cells can be appropriately reduced (15-30 min).</li>
	<li>Wash the cells with 1x PBS (1 mL/well) three times. Add 1 mL of 1x PBS to each well.</li>
	<li>Place the 12-well plate on the microscope stage (see Table of Materials), and then use a 20x objective to observe the morphology of the cells (magnification: 200x).</li>
	<li>Capture three representative fluorescence images (magnification: 200x) for each well on the fluorescence microscope using the green fluorescent channel with an excitation wavelength of 480 nm and an emission wavelength of 530 nm. 
	NOTE: The green fluorescent channel with an excitation wavelength of 480 nm and an emission wavelength of 530 nm is recommended to detect the DCFH-DA. Additionally, DCFH-DA can be detected with the parameter settings for GFP and FITC in the fluorescence microscope35,36.</li>
	<li>Finally, process the images with image-acquisition software (see Table of Materials), and then use ImageJ software to calculate the mean fluorescence intensity of each group or the ratios of the different groups. 
	&#8203;NOTE: The technical details of the fluorescence microscope and Image J software used for the imaging work have been described previously37,38.</li>
</ol>
3. Measurement of the change in mitochondrial membrane potential
NOTE: The changes in mitochondrial membrane potential are monitored by the JC-1 staining assay.
<ol>
	<li>At the end of the treatment period (step 1.8), wash the cells with 1 mL of 1x PBS per well three times, and then stain the cells with 100 &#181;L of 5 &#181;g/mL JC-1 working solution (see Table of Materials) for 30 min at 37 &#176;C in the dark. 
	NOTE: If no obvious green fluorescence is observed after 30 min of incubation, the incubation time of the probe and cells can be appropriately increased (30-60 min). If overexposure of the green fluorescence value is observed after 30 min of incubation, the incubation time of the probe and cells can be appropriately reduced (15-30 min).</li>
	<li>Wash the cells with 1x PBS (1 mL/well) three times. Add 1 mL of 1x PBS to each well.</li>
	<li>Place the 12-well plate on the microscope stage, and then use a 20x objective to observe the morphology of the cells (magnification: 200x).</li>
	<li>Capture three representative fluorescence images (magnification: 200x) for each well on the fluorescence microscope using the green fluorescent channel with excitation and emission wavelengths of 485 nm and 535 nm, respectively, and the red fluorescent channel with excitation and emission wavelengths of 550 nm and 600 nm, respectively. 
	NOTE: The green fluorescent channel with an excitation wavelength of 485 nm and an emission wavelength of 535 nm is used to detect the JC-1 monomer, which is treated as depolarized mitochondria39,40,41, and the red fluorescent channel with an excitation wavelength of 550 nm and an emission wavelength of 600 nm is used to detect the JC-1 dimer, which is treated as polarized mitochondria39,40,41.</li>
	<li>Finally, process the images with image-acquisition software, and then use Image J software to calculate the mean fluorescence intensity of each group or the ratios of the different groups. 
	&#8203;NOTE: The technical details of the fluorescence microscope and Image J software used for the imaging work have been described previously37,38.</li>
</ol>
4. Measurement of the protein expression levels of LC3-II/LC3-I and p62/SQSTM1
<ol>
	<li>After treatment (step 1.8), wash the cells three times with 1x PBS (1 mL/well) that has been precooled at 4 &#176;C.</li>
	<li>Add RIPA lysis buffer (100 &#181;L/well) supplemented with protease and a phosphatase inhibitor cocktail (1x) (see Table of Materials) to the 12-well plate, and lyse on ice for 5 min.</li>
	<li>Collect the cell lysate into 1.5 mL microcentrifuge tubes, and centrifuge at 12,000 x g for 20 min at 4 &#176;C. Determine the protein concentration of the supernatants by the BCA method following the standard procedures42.</li>
	<li>Add SDS-PAGE sample loading buffer (5x, see Table of Materials) to the cell lysate supernatants (volume ratio = 1:4).</li>
	<li>Mix by vortexing (at high speed for ~15 s), and heat the mixed samples at 100 &#176;C for 5 min to denature the protein.</li>
	<li>Put the 12-well-prepared 12% SDS-PAGE gel into the electrophoresis tank, and then add the SDS up-sample buffer (1x) diluted with ultra-pure water to the height limit position. 
	NOTE: The SDS-PAGE gel is prepared using a commercial kit (see Table of Materials) according to the manufacturer&#39;s instructions.</li>
	<li>Add the protein marker (5 &#181;L/well) and samples (20 &#181;g/well) into different wells of the SDS-PAGE gel.</li>
	<li>Set the stable voltage mode at 100 V, and perform the electrophoresis for 80 min.</li>
	<li>After the SDS-PAGE electrophoresis, carry out the electrotransfer of the proteins to a polyvinylidene fluoride (PVDF) membrane (0.45 &#181;M, see Table of Materials) following previously published reports43,44.</li>
	<li>After the electrotransfer of the proteins, wash the PVDF membrane with 10 mL of TBST (1x TBS, 0.1% Tween 20) two times (5 min/time) in a shaker at room temperature.</li>
	<li>Block the PVDF membrane with 5 mL of bovine serum albumin (BSA, 5%) in a shaker at room temperature for 1 h.</li>
	<li>Wash the PVDF membrane with 10 mL of TBST three times (10 min/time). Then, incubate the PVDF membrane in 5 mL of the specific primary antibodies diluted in blocking buffer for LC3 (mouse mAb, 1:2,000), p62/SQSTM1 (hereafter referred to as p62, mouse mAb, 1:2,000), and &#946;-actin (mouse mAb, 1:2,000) (see Table of Materials) overnight at 4 &#176;C.</li>
	<li>Wash the PVDF membrane with 10 mL of TBST three times (10 min/time) at room temperature. Then, incubate the PVDF membrane with rabbit anti-mouse IgG (HRP) secondary antibody diluted in blocking buffer (1:10,000) (see Table of Materials) at room temperature and sheltered from light for 2 h.</li>
	<li>Wash the PVDF membrane with 10 mL of TBST three times (10 min/time) at room temperature. Then, place the PVDF membrane on the plastic wrap, add an appropriate amount of ECL working solution (200 &#181;L/membrane) (see Table of Materials), and incubate for 2 min.</li>
	<li>After incubation, remove the ECL working solution, and expose the PVDF membrane in the imaging system for image development. Finally, use the Image J software to analyze the gray values of each band. 
	&#8203;NOTE: The technical details of the western blotting and Image J software used for the imaging work have been described previously45,46.</li>
</ol>
5. Statistical analysis
<ol>
	<li>Present the data from the experiments as the mean &#177; standard deviation (SD).</li>
	<li>Perform the analysis of significance with the statistical software tool described previously47.</li>
	<li>Calculate the statistical differences between the two groups using a t-test. A P-value below 0.05 is considered statistically significant: *P &#60; 0.05, **P &#60; 0.01, ***P &#60; 0.005.</li>
</ol>

The authors declare no conflicts of interest.

Studies have highlighted the fact that NAFLD is a clinicopathological syndrome, ranging from fatty liver to NASH, that can progress to cirrhosis and liver cancer51. A high-fat diet and an inactive lifestyle are typical risk factors for NAFLD. Both non-drug therapies and drug therapies for NAFLD treatment have been researched51,52,53. However, the pathogenesis of NAFLD has not been fully elucidated. The me...

Platycodon grandiflorus (PG), which is the dried root of Platycodon grandiflorus (Jacq.) A.DC., is used in traditional Chinese medicine (TCM). It is mainly produced in the northeast, north, east, central, and southwest regions of China1. The PG components include triterpenoid saponins, polysaccharides, flavonoids, polyphenols, polyethylene glycols, volatile oils, and minerals2. PG has a long history of being used as a food and a herbal medicine in Asia. Traditionally, this herb was used to make medicine against lung diseases. Modern pharmacology also provides evidence of the efficacy of PG for treating other diseases. Studies have shown that PG has a therapeutic effect on a variety of drug-induced liver injury models. The dietary supplementation of PG or platycodin extracts can ameliorate high-fat diet-induced obesity and its related metabolic diseases3,4,5. Polysaccharides from PG can be used for the treatment of acute liver injury caused by LPS/D-GalN in mice6. Moreover, saponins from the roots of PG ameliorate high-fat diet-induced non-alcoholic steatohepatitis (NASH)7. Furthermore, platycodin D (PD), one of the most important therapeutic components of PG, can enhance low-density lipoprotein receptor expression and low-density lipoprotein uptake in human hepatocellular carcinoma (HepG2) cells8. Furthermore, PD can also induce apoptosis and inhibit adhesion, migration, and invasion in HepG2 cells9,10. Thus, in this study, mouse hepatoma AML-12 cells are used for in vitro model construction and to further study the pharmacological effects and underlying mechanisms of PD in this model.
The term non-alcoholic fatty liver disease (NAFLD) refers to a group of liver diseases that includes simple steatosis, NASH, cirrhosis, and hepatocellular carcinoma11. Although the pathogenesis of NAFLD is incompletely understood, from the classic &#34;two-hit&#34; theory to the current &#34;multiple-hit&#34; theory, insulin resistance is considered to be central in the pathogenesis of NAFLD12,13,14. Studies have demonstrated that insulin resistance in hepatocytes could lead to increased free fatty acids, which form triglycerides that are deposited in the liver and cause the liver to become fatty15,16. The accumulation of fat can lead to lipotoxicity, oxidative stress-induced mitochondrial dysfunction, endoplasmic reticulum stress, and inflammatory cytokine release, resulting in the pathogenesis and progression of NAFLD17,18. In addition, autophagy also plays a role in the pathogenesis of NAFLD, as it is involved in regulating cellular insulin sensitivity, cellular lipid metabolism, hepatocyte injury, and innate immunity19,20,21.
A variety of animal models and cellular models have been established to provide a basis for exploring the pathogenesis and potential therapeutic targets of NAFLD22,23. However, single animal models cannot fully mimic all the pathological processes of NAFLD24. Individual differences between animals lead to different pathological features. Using liver cell lines or primary hepatocytes in in vitro studies of NAFLD ensures maximum consistency in the experimental conditions. Hepatic lipid metabolism dysregulation can lead to higher levels of hepatocyte lipid droplet accumulation in NAFLD25. Free fatty acids such as oleic acid and palm oil have been used in the in vitro model to mimic NAFLD caused by a high-fat diet26,27. The human hepatoblastoma cell line HepG2 is often used in the construction of NAFLD models in vitro, but, as a tumor cell line, the metabolism of HepG2 cells is significantly different from that of liver cells under normal physiological conditions28. Therefore, using primary hepatocytes or mouse primary hepatocytes to construct the in vitro NAFLD model for drug screening is more advantageous than using tumor cell lines. Comparing the synergistic examination of drug effects and therapeutic targets in both animal models and in vitro hepatocyte models, it seems that using mouse hepatocytes to construct the in vitro NAFLD model has better application potential.
Free fatty acids entering the liver are oxidized to produce energy or stored as triglycerides. Significantly, free fatty acids have a certain lipotoxicity and may induce cellular dysfunction and apoptosis12. Palmitic acid (PA) is the most abundant saturated fatty acid in human plasma29. When cells in non-adipose tissue are exposed to high concentrations of PA for a long time, this stimulates the production of reactive oxygen species (ROS) and causes oxidative stress, lipid accumulation, and even apoptosis30. Therefore, many researchers use PA as an inducer to stimulate liver cells to produce ROS and, thus, construct the in vitro fatty liver disease model and evaluate the protective effects of certain active substances on cells31,32,33,34. This study introduces a protocol for investigating the protective effects of PD on a cell model of NAFLD induced by PA.

<table><tbody><tr><td>5% BSA Blocking Buffer</td><td>Solarbio, Beijing, China</td><td>SW3015</td><td></td></tr><tr><td>AML12 (alpha mouse liver 12) cell line</td><td>Procell Life Science&amp;amp;Technology Co., Ltd, China</td><td>AML12</td><td></td></tr><tr><td>Beyo ECL&amp;nbsp;Plus</td><td>Beyotime, Shanghai, China</td><td>P0018S</td><td></td></tr><tr><td>Bio-safety cabinet</td><td>Esco Micro Pte Ltd, Singapore</td><td>AC2-5S1 A2&amp;nbsp;</td><td></td></tr><tr><td>cellSens</td><td>Olympus, Tokyo, Japan</td><td>1.8</td><td></td></tr><tr><td>Culture CO2 Incubator</td><td>Esco Micro Pte Ltd, Singapore</td><td>CCL-170B-8</td><td></td></tr><tr><td>Dexamethasone</td><td>Beyotime, Shanghai, China</td><td>ST125</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Solarbio, Beijing, China</td><td>D8371</td><td></td></tr><tr><td>DMEM/F12</td><td>Hyclone,&amp;nbsp;Logan,&amp;nbsp;UT,&amp;nbsp;USA</td><td>SH30023.01</td><td></td></tr><tr><td>Foetal Bovine Serum</td><td>Hyclone, Tauranga, New Zealand</td><td>SH30406.05</td><td></td></tr><tr><td>Graphpad software</td><td>GraphPad Software Inc., San Diego, CA, USA</td><td>8.0</td><td></td></tr><tr><td>HRP Goat Anti-Mouse IgG (H+L)</td><td>ABclonal, Wuhan, China</td><td>AS003</td><td></td></tr><tr><td>Hydrophobic PVDF Transfer Membrane</td><td>Merck, Darmstadt, Germany</td><td>IPFL00010</td><td></td></tr><tr><td>Insulin, Transferrin, Selenium Solution, 100&amp;times;</td><td>Beyotime, Shanghai, China</td><td>C0341</td><td></td></tr><tr><td>MAP LC3&amp;beta; Antibody</td><td>Santa Cruz Biotechnology (Shanghai) Co., Ltd</td><td>SC-376404</td><td></td></tr><tr><td>Mitochondrial Membrane Potential Assay Kit with JC-1</td><td>Solarbio, Beijing, China</td><td>M8650</td><td></td></tr><tr><td>Olympus Inverted Microscope IX53</td><td>Olympus, Tokyo, Japan</td><td>IX53</td><td></td></tr><tr><td>Palmitic Acid</td><td>Sigma, Germany</td><td>P0500</td><td></td></tr><tr><td>Penicillin-Streptomycin Solution (100x)</td><td>Hyclone,&amp;nbsp;Logan,&amp;nbsp;UT,&amp;nbsp;USA</td><td>SV30010</td><td></td></tr><tr><td>Phenylmethanesulfonyl&amp;nbsp;fluoride</td><td>Beyotime, Shanghai, China</td><td>ST506</td><td></td></tr><tr><td>Phosphate Buffered Solution</td><td>Hyclone,&amp;nbsp;Logan,&amp;nbsp;UT,&amp;nbsp;USA</td><td>BL302A</td><td></td></tr><tr><td>Platycodin D</td><td>Chengdu Must Bio-Technology Co., Ltd, China</td><td>CSA: 58479-68-8</td><td></td></tr><tr><td>Protease inhibitor cocktail for general use, 100x</td><td>Beyotime, Shanghai, China</td><td>P1005</td><td></td></tr><tr><td>Protein Marker</td><td>Solarbio, Beijing, China</td><td>PR1910</td><td></td></tr><tr><td>Reactive Oxygen Species Assay Kit</td><td>Solarbio, Beijing, China</td><td>CA1410</td><td></td></tr><tr><td>RIPA&amp;nbsp;Lysis&amp;nbsp;Buffer</td><td>Beyotime, Shanghai, China</td><td>P0013E</td><td></td></tr><tr><td>SDS-PAGE Gel Quick Preparation Kit</td><td>Beyotime, Shanghai, China</td><td>P0012AC</td><td></td></tr><tr><td>SDS-PAGE Sample Loading Buffer, 5x</td><td>Beyotime, Shanghai, China</td><td>P0015</td><td></td></tr><tr><td>Sigma Centrifuge</td><td>Sigma, Germany</td><td>3K15</td><td></td></tr><tr><td>SQSTM1/p62 Antibody</td><td>Santa Cruz Biotechnology (Shanghai) Co., Ltd</td><td>SC-28359</td><td></td></tr><tr><td>Tecan Infinite 200 PRO&amp;nbsp;&amp;nbsp;</td><td>Tecan Austria GmbH, Austria</td><td>1510002987</td><td></td></tr><tr><td>WB Transfer Buffer,10x</td><td>Solarbio, Beijing, China</td><td>D1060</td><td></td></tr><tr><td>&amp;beta;-Actin Mouse mAb</td><td>ABclonal, Wuhan, China</td><td>AC004</td><td></td></tr></tbody></table>

investigating the protective effects of platycodin d on non-alcoholic fatty liver disease in a palmitic acid-induced in vitro model

The occurrence of non-alcoholic fatty liver disease (NAFLD) has been increasing at an alarming rate worldwide. Platycodon grandiflorum is widely used as a traditional ethnomedicine for the treatment of various diseases and is a typical functional food that can be incorporated into the everyday diet. Studies have suggested that platycodin D (PD), one of the main active ingredients in Platycodon grandiflorum, has high bioavailability and significantly mitigates the progress of NAFLD, but the underlying mechanism of this is still unclear. This study aims to investigate the therapeutic effect of PD against NAFLD in vitro. AML-12 cells were pretreated with 300 &#181;M palmitic acid (PA) for 24 h to model NAFLD in vitro. Then, the cells were either treated with PD or received no PD treatment for 24 h. The levels of reactive oxygen species (ROS) were analyzed using 2&#8242;,7&#8242;-dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining, and the mitochondrial membrane potential was determined by the JC-1 staining method. Moreover, the protein expression levels of LC3-II/LC3-I and p62/SQSTM1 in the cell lysates were analyzed by western blotting. PD was found to significantly decrease the ROS and mitochondrial membrane potential levels in the PA-treated group compared to the control group. Meanwhile, PD increased the LC3-II/LC3-I levels and decreased the p62/SQSTM1 levels in the PA-treated group compared to the control group. The results indicated that PD ameliorated NAFLD in vitro by reducing oxidative stress and stimulating autophagy. This in vitro model is a useful tool for studying the role of PD in NAFLD.

Intracellular ROS in the cells 
AML-12 cells were induced with 300 &#181;M PA for 24 h, and a NAFLD cell model was established. Subsequently, the cells were treated with PD for 24 h. The cells were labeled with a DCFH-DA fluorescent probe, and ROS production was observed under a fluorescence microscope. The results of the DCFH-DA staining of intracellular ROS in the cells are shown in Figure 1. The results showed that PD could significantly reduce the level of intracell...

Watch this Scientific Journal Video about Investigating the Protective Effects of Platycodin D on Non-Alcoholic Fatty Liver Disease in a Palmitic Acid-Induced In Vitro Model at JoVE.com

Investigating the Protective Effects of Platycodin D on Non-Alcoholic Fatty Liver Disease in a Palmitic Acid-Induced In Vitro Model

This protocol investigates the protective effects of platycodin D on non-alcoholic fatty liver disease in a palmitic acid-induced in vitro model.

Investigating the Protective Effects of Platycodin D on Non-Alcoholic Fatty Liver Disease in a Palmitic Acid-Induced In Vitro Model

The occurrence of non-alcoholic fatty liver disease (NAFLD) has been increasing at an alarming rate worldwide. Platycodon grandiflorum is widely used as a ...

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1.8K Views.  Chongqing Academy of Chinese Materia Medica. This protocol investigates the protective effects of platycodin D on non-alcoholic fatty liver disease in a palmitic acid-induced in vitro model.

Video: Investigating the Protective Effects of Platycodin D on Non-Alcoholic Fatty Liver Disease in a Palmitic Acid-Induced In Vitro Model

Measurement of Fatty Acid &#946;-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional processes, such as ketogenesis and gluconeogenesis, which are essential to maintain whole-body glucose homeostasis and support extra-hepatic organ function in the fasted state. Fatty acid &#946;-oxidation occurs within the mitochondria and peroxisomes and is regulated through multiple mechanisms, including the uptake and activation of fatty acids, enzyme expression levels, and availability of cofactors such as coenzyme A and NAD+. In assays that measure fatty acid &#946;-oxidation in liver homogenates, cell lysis and the common addition of supraphysiological levels of cofactors mask the effects of these regulatory mechanisms. Furthermore, the integrity of the organelles in the homogenates is hard to control and can vary significantly between preparations. The measurement of fatty acid &#946;-oxidation in intact primary hepatocytes overcomes the above pitfalls. This protocol describes a method for the measurement of fatty acid &#946;-oxidation in a suspension of freshly isolated primary mouse hepatocytes incubated with 14C-labeled palmitic acid. By avoiding hours to days of culture, this method has the advantage of better preserving the protein expression levels and metabolic pathway activity of the original liver, including the activation of fatty acid &#946;-oxidation observed in hepatocytes isolated from fasted mice compared to fed mice.

Fatty acid &#946;-oxidation is an essential metabolic pathway responsible for generating energy in many different cell types, including hepatocytes. Here, we describe a method to measure fatty acid &#946;-oxidation in freshly isolated primary hepatocytes using 14C-labeled palmitic acid.

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional ...

Biochemistry

Assessing Hepatic Steatosis with 3D Fluorescence Lipid Droplet Reconstruction

Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment

Lipid droplets (LDs) are specialized organelles that mediate lipid storage and play a very important role in suppressing lipotoxicity and preventing dysfunction caused by free fatty acids (FAs). The liver, given its critical role in the body's fat metabolism, is persistently threatened by the intracellular accumulation of LDs in the form of both microvesicular and macrovesicular hepatic steatosis. The histologic characterization of LDs is typically based on lipid-soluble diazo dyes, such as Oil Red O (ORO) staining, but a number of disadvantages consistently hamper the use of this analysis with liver specimens. More recently, lipophilic fluorophores 493/503 have become popular for visualizing and locating LDs due to their rapid uptake and accumulation into the neutral lipid droplet core. Even though most applications are well-described in cell cultures, there is less evidence demonstrating the reliable use of lipophilic fluorophore probes as an LD imaging tool in tissue samples. Herein, we propose an optimized boron dipyrromethene (BODIPY) 493/503-based protocol for the evaluation of LDs in liver specimens from an animal model of high-fat diet (HFD)-induced hepatic steatosis. This protocol covers liver sample preparation, tissue sectioning, BODIPY 493/503 staining, image acquisition, and data analysis. We demonstrate an increased number, intensity, area ratio, and diameter of hepatic LDs upon HFD feeding. Using orthogonal projections and 3D reconstructions, it was possible to observe the full content of neutral lipids in the LD core, which appeared as nearly spherical droplets. Moreover, with the fluorophore BODIPY 493/503, we were able to distinguish microvesicles (1 &#181;m &lt; d &#8804; 3 &#181;m), intermediate vesicles (3 &#181;m &lt; d &#8804; 9 &#181;m), and macrovesicles (d &gt; 9 &#181;m), allowing the successful discrimination of microvesicular and macrovesicular steatosis. Overall, this BODIPY 493/503 fluorescence-based protocol is a reliable and simple tool for hepatic LD characterization and may represent a complementary approach to the classical histological protocols.

Author Spotlight: Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment  

Herein, we demonstrate an optimized BODIPY 493/503 fluorescence-based protocol for lipid droplet characterization in liver tissue. Through the use of orthogonal projections and 3D reconstructions, the fluorophore allows for successful discrimination between microvesicular and macrovesicular steatosis and may represent a complementary approach to the classical histological protocols for hepatic steatosis assessment.

Lipid droplets (LDs) are specialized organelles that mediate lipid storage and play a very important role in suppressing lipotoxicity and preventing ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders.
In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via &#946;-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying &#946;-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. 
Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of &#946;-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.

De novo lipogenesis and &#946;-fatty acid oxidation constitute key metabolic pathways in hepatocyte, pathways that are perturbed in several metabolic disorders, including fatty liver disease. Here we demonstrate isolation of mouse primary hepatocytes and describe quantification of &#946;-fatty acid oxidation and lipogenesis.

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid ...

Biology

A Model of Experimental Steatosis In Vitro: Hepatocyte Cell Culture in Lipid Overload-Conditioned Medium

Metabolic dysfunction-associated fatty liver disease (MAFLD), previously known as non-alcoholic fatty liver disease (NAFLD), is the most prevalent liver disease worldwide due to its relationship with obesity, diabetes type 2, and dyslipidemia. Hepatic steatosis, the accumulation of lipid droplets in the liver parenchyma, is a key feature of the disease preceding the inflammation observed in steatohepatitis, fibrosis, and end-stage liver disease. Lipid accumulation in hepatocytes might interfere with proper metabolism of xenobiotics and endogenous molecules, as well as to induce cellular processes leading to the advance of the disease. Although the experimental study of steatosis can be performed in vivo, in vitro approaches to the study of steatosis are complementary tools with different advantages. Hepatocyte culture in lipid overload-conditioned medium is an excellent reproducible option for the study of hepatic steatosis allowing the identification of cellular processes related to lipid accumulation, such as oxidative and reticular stresses, autophagia, proliferation, cell death, etcetera, as well as other testing including drug effectiveness, and toxicological testing, among many other possible applications. Here, it was aimed to describe the methodology of hepatocyte cell culture in lipid overload-conditioned medium. HepG2 cells were cultured in RMPI 1640 medium conditioned with sodium palmitate and sodium oleate. Importantly, the ratio of these two lipids is crucial to favor lipid droplet accumulation, while maintaining cell proliferation and a moderate mortality rate, as occurs in the liver during the disease. The methodology, from the preparation of the lipid solution stocks, mixture, addition to the medium, and hepatocyte culture is shown. With this approach, it is possible to identify lipid droplets in the hepatocytes that are readily observable by Oil-red O staining, as well as curves of proliferation/mortality rates.

A Model of Experimental Steatosis In Vitro: Hepatocyte Cell Culture in Lipid Overload-Conditioned Medium

This protocol is intended to be a tool to study steatosis and the molecular, biochemical, cellular changes produced by the over exposure of hepatocytes to lipids in vitro.

Metabolic dysfunction-associated fatty liver disease (MAFLD), previously known as non-alcoholic fatty liver disease (NAFLD), is the most prevalent liver ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...

The Murine Choline-Deficient, Ethionine-Supplemented (CDE) Diet Model of Chronic Liver Injury

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual inflammation, progressive destruction and regeneration of the hepatic parenchyma, liver progenitor cell proliferation, and fibrosis. The end-stage of every chronic liver disease is cirrhosis, a major risk factor for the development of hepatocellular carcinoma. To study processes regulating disease initiation, establishment, and progression, several animal models are used in laboratories. Here we describe a six-week time course of the choline-deficient and ethionine-supplemented (CDE) mouse model, which involves feeding six-week old male C57BL/6J mice with choline-deficient chow and 0.15% DL-ethionine-supplemented drinking water. Monitoring of animal health and a typical body weight loss curve are explained. The protocol demonstrates the gross examination of a CDE-treated liver and blood collection by cardiac puncture for subsequent serum analyses. Next, the liver perfusion technique and collection of different hepatic lobes for standard evaluations are shown, including liver histology assessments by hematoxylin and eosin or Sirius Red stainings, immunofluorescent detection of hepatic cell populations as well as transcriptome profiling of the liver microenvironment. This mouse model is suitable for studying inflammatory, fibrogenic, and liver progenitor cell dynamics induced through chronic liver disease and can be used to test potential therapeutic agents that may modulate these processes.

The Murine Choline-Deficient, Ethionine-Supplemented CDE Diet Model of Chronic Liver Injury

Here we describe a common method to induce chronic liver injury in mice by feeding of a choline-deficient and ethionine-supplemented (CDE) diet. We demonstrate health monitoring, liver perfusion, isolation, and preservation. A time course of six weeks can inform about liver injury, pathohistology, fibrosis, inflammatory, and liver progenitor cell responses.

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual ...

Network Pharmacology Prediction and Metabolomics Validation of the Mechanism of Fructus Phyllanthi against Hyperlipidemia

Hyperlipidemia has become a leading risk factor for cardiovascular diseases and liver injury worldwide. Fructus Phyllanthi (FP) is an effective drug against hyperlipidemia in Traditional Chinese Medicine (TCM) and Indian Medicine theories, however the potential mechanism requires further exploration. The present research aims to reveal the mechanism of FP against hyperlipidemia based on an integrated strategy combining network pharmacology prediction with metabolomics validation. A high-fat diet (HFD)-induced mice model was established by evaluating the plasma lipid levels, including total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C). Network pharmacology was applied to find out the active ingredients of FP and potential targets against hyperlipidemia. Metabolomics of plasma and liver were performed to identify differential metabolites and their corresponding pathways among the normal group, model group, and intervention group. The relationship between network pharmacology and metabolomics was further constructed to obtain a comprehensive view of the process of FP against hyperlipidemia. The obtained key target proteins were verified by molecular docking. These results reflected that FP improved the plasma lipid levels and liver injury of hyperlipidemia induced by a HFD. Gallic acid, quercetin, and beta-sitosterol in FP were demonstrated as the key active compounds. A total of 16 and six potential differential metabolites in plasma and liver, respectively, were found to be involved in the therapeutic effects of FP against hyperlipidemia by metabolomics. Further, integration analysis indicated that the intervention effects were associated with CYP1A1, AChE, and MGAM, as well as the adjustment of L-kynurenine, corticosterone, acetylcholine, and raffinose, mainly involving tryptophan metabolism pathway. Molecular docking ensured that the above ingredients acting on hyperlipidemia-related protein targets played a key role in lowering lipids. In summary, this research provided a new possibility for preventing and treating hyperlipidemia.

The present protocol describes an integrated strategy for exploring the key targets and mechanisms of Fructus Phyllanthi against hyperlipidemia based on network pharmacology prediction and metabolomics verification.

Hyperlipidemia has become a leading risk factor for cardiovascular diseases and liver injury worldwide. Fructus Phyllanthi (FP) is an effective drug ...

Live Cell Imaging of Lipid Droplet Size and Fusion Using Oil Red O and BODIPY

Evaluation of Lipid Droplet Size and Fusion in Bovine Hepatic Cells

Lipid droplets (LDs) are organelles that play an important role in lipid metabolism and neutral lipid storage in cells. They are associated with a variety of metabolic diseases, such as obesity, fatty liver disease, and diabetes. In hepatic cells, the sizes and numbers of LDs are signs of fatty liver disease. Moreover, the oxidative stress reaction, cell autophagy, and apoptosis are often accompanied by changes in the sizes and numbers of LDs. As a result, the dimensions and quantity of LDs are the basis of the current research regarding the mechanism of LD biogenesis. Here, in fatty acid-induced bovine hepatic cells, we describe how to use oil red O to stain LDs and to investigate the sizes and numbers of LDs. The size distribution of LDs is statistically analyzed. The process of small LDs fusing into large LDs is also observed by a live cell imaging system. The current work provides a way to directly observe the size change trend of LDs under different physiological conditions.

Author Spotlight: Evaluation of Lipid Droplet Size and Fusion in Bovine Hepatic Cells  

The present protocol describes how to use oil red O to dye lipid droplets (LDs), calculate the size and number of LDs in a fatty acid-induced fatty hepatocyte model, and use BODIPY 493/503 to observe the process of small LDs fusing into large LDs by live cell imaging.

Lipid droplets (LDs) are organelles that play an important role in lipid metabolism and neutral lipid storage in cells. They are associated with a variety ...

Investigating the Protective Effects of Platycodin D on Non-Alcoholic Fatty Liver Disease in a Palmitic Acid-Induced In Vitro Model

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Measurement of Fatty Acid β-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes

Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

A Model of Experimental Steatosis In Vitro: Hepatocyte Cell Culture in Lipid Overload-Conditioned Medium

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

The Murine Choline-Deficient, Ethionine-Supplemented (CDE) Diet Model of Chronic Liver Injury

Network Pharmacology Prediction and Metabolomics Validation of the Mechanism of Fructus Phyllanthi against Hyperlipidemia

Evaluation of Lipid Droplet Size and Fusion in Bovine Hepatic Cells