Name: Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis (Video) | JoVE Journal
Uploaded: 2017-03-11T15:00:00.000+00:00
Duration: 8 min 58 s
Description: Learn optimized methods to generate and characterize hepatic steatosis using in vivo and in vitro approaches. These techniques enable noninvasive assessment of liver lipid metabolism and disease progression.

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5 minutes

We appreciate Feifei Zhang for the helpful discussions. We are grateful to Jing Gao and Yixuan Sun for the technical assistance and to Zhengshuai Liu and Fengguang Ma for the animal studies.

All animal experimental protocols were approved by the institutional animal care and use committee at the Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China).
1. DIO Mouse Model
<ol>
	<li>HFHS feeding
	<ol>
		<li>Feed the eight-week-old male C57BL/6 mice with an HFHS that contains 40 kcal% fat and 40 kcal% sucrose. House them in 12 h dark-light cycle conditions.</li>
		<li>Keep the mice in HFHS diet-feeding condition for four to sixteen weeks. Check the body weight weekly.</li>
	</ol>
	</li>
	<li>Body composition analysis and pseudo-color image analysis
	<ol>
		<li>Subject the mice to a body composition analyzer analysis.
		<ol>
			<li>Secure conscious, not anesthetized mice in a plastic tube in the. Measure the body fat mass, lean mass, and fluids using an NMR system, as described previously2.</li>
		</ol>
		</li>
		<li>Scan the whole body of each mouse using an MRI system.
		<ol>
			<li>Anesthetize the mice with 1% avertin via intraperitoneal injection (25 &#181;L/g) and perform the imaging analysis in specialized glass tubes. 
			NOTE: The process of imaging lasts for nearly 0.5 h for each mouse. When the measurement is completed, return the mice to their cage.</li>
		</ol>
		</li>
		<li>Scan the whole bodies of the mice in dorsal, middle, and ventral layers; scan the liver in the vertical direction. Use a 0.55 T magnet and the following instrumental parameters: K space = 192 x 256 &#181;m, section thickness = 3 mm, TE = 14.8 ms, TR = 400 ms, FOVRead = 100 mm, FOVPhase = 100 mm, and number of scans = 8. Scan using the same magnetic condition for the all groups of mice.</li>
		<li>Map the grayscale image onto a pseudo-color image according to the signal intensity9.</li>
	</ol>
	</li>
	<li>Mouse euthanasia
	<ol>
		<li>Sterilize dissecting tools, such as forceps and scissors.</li>
		<li>Pour sufficient isoflurane on a paper towel and let the isoflurane volatilize in a glass jar. Ensure that the mice are euthanized by the isoflurane.</li>
		<li>Fix the mice on an operating table. Spray each abdomen with 75% ethanol, make an incision in the dermis of the xiphoid process using scissors, and tear the dermis lengthwise through the midline with the hands to expose the peritoneum.</li>
		<li>Clamp the xiphoid process with forceps and cut the peritoneum breadthwise through the bottom of the ribs to expose the viscera.</li>
		<li>Collect blood in EDTA-coated tubes by cardiac puncture.</li>
		<li>Cut the diaphragm along the liver and carefully remove the liver from the enterocoelia using fine scissors. Cut three pieces of liver tissue for the following lipid measurements: a) 6 x 3 x 3 mm for H&#38;E staining, b) 6 x 3 x 3 mm for Oil Red O staining, and c) 20-40 mg of liver for chloroform/methanol extraction.</li>
	</ol>
	</li>
	<li>H&#38;E staining
	<ol>
		<li>Fix the liver in 4% phosphate-buffered formalin acetate at 4 &#176;C overnight and then embed it in paraffin wax.</li>
		<li>Cut paraffin sections (5 &#181;m) and mount them on glass slides for H&#38;E staining, as previously described3,10.</li>
	</ol>
	</li>
	<li>Oil Red O staining
	<ol>
		<li>Regent preparation
		<ol>
			<li>Prepare Oil Red O working solution with 10% formalin, 60% isopropanol, Hematoxylin, and glycerol jelly. Dissolve 0.5 g of Oil Red O powder with 100 mL of isopropanol and heat it in a 60 &#176;C bath to make a 0.5% stock solution.</li>
			<li>Dilute with double distilled water (ddH2O) at a 3:2 ratio (3 mL of stock solution and 2 mL of ddH2O). Filter the working solution and let stand for at least 10 min at room temperature.</li>
		</ol>
		</li>
		<li>Embed the liver in optimum cutting temperature (OCT) compound in a plastic embedding box. Freeze it at -20 &#176;C for 30 min. Cut it into 8-&#181;m sections in a freezing microtome, mount the tissue on a slide, and place it at room temperature for 30 min.</li>
		<li>Place a paper towel in the bottom of a staining tub and pour a small amount of 10% formalin to wet the paper towel. Place the slide in the staining tub and fix the frozen sections in formaldehyde vapors for 5 min at 4 &#176;C.</li>
		<li>Dip the slide in 60% isopropanol in the staining tub for 3 s; repeat once.</li>
		<li>Add a few drops of Oil Red O working solution to the slide to cover all tissue and incubate for 15 min. Protect the tissue from light at room temperature.</li>
		<li>Rinse the slide in new 60% isopropanol in the staining tub for 3 s; repeat twice.</li>
		<li>Gently rinse the slide through distilled water twice and remove the water around the tissue. Be sure not to dry the tissue.</li>
		<li>Place the slide in Hematoxylin for 1 min and rinse gently with tap water for 5-10 min. Gently rinse the slide in distilled water twice and keep the it in distilled water until ready to put on the coverslip.</li>
		<li>Heat up the glycerol jelly in the microwave and place several drops on top of the tissue. Gently place the coverslip on top and take care not to form bubbles.</li>
		<li>Observe the stain under a microscope and capture characteristic pictures using 20X and 40X objectives.</li>
	</ol>
	</li>
	<li>Measurement of liver lipids
	<ol>
		<li>Place the dissected liver tissue in a new 2-mL plastic centrifuge tube. Homogenize in 1 mL of PBS with a homogenizer and transfer the lysate to a new 15-mL glass tube.</li>
		<li>Add 5 mL of chloroform/methanol (2:1, v/v) solution, vortex the mixture vigorously for 1 min, and let stand for 2 h on ice.</li>
		<li>Centrifuge at 1,650 x g for 10 min at 4 &#176;C; the mixture should separate into 3 layers: the upper methanol layer, the middle protein disc, and the bottom chloroform and lipids.</li>
		<li>Transfer the bottom phase into a new glass tube using a glass Pasteur pipet; there is no need to get every drop of the bottom fraction. Set the tube aside on ice.</li>
		<li>Add 600 &#181;L of 4 mM MgCl2 and 1.5 mL of chloroform to the supernatant fraction left in the previous tube and vortex vigorously. Incubate on ice for 30 min and centrifuge at 1,650 x g for 10 min at 4 &#176;C.</li>
		<li>Combine the bottom phase with the first bottom phase tube using Pasteur pipet. Discard the upper and middle layers.</li>
		<li>Evaporate the chloroform under nitrogen gas in a 37 &#176;C bath. Wash the sides of the glass tubes thoroughly with 200 &#181;L of 1% Triton X-100/chloroform (v/v). Evaporate under nitrogen gas. Dissolve the lipid with 200 &#181;L of ddH2O and vortex well to make the final emulsion.</li>
		<li>Measure the total triglyceride (TG) and cholesterol (TC) levels by using a triglyceride or cholesterol kit according to the manufacturer&#39;s protocol3. 
		NOTE: The lipid values are normalized to those of the protein concentrations in the initial homogenate, as determined by protein quantification3.</li>
	</ol>
	</li>
</ol>
2. Primary Mouse Hepatocyte Model
<ol>
	<li>Isolation of mouse primary hepatocytes
	<ol>
		<li>Anesthetize each mouse with 6% chloral hydrate (10 &#181;L/g intraperitoneally).</li>
		<li>Isolate primary hepatocytes, as previously described10. Grow cells on collagen-coated glass coverslips in a 6-well plate for Oil Red O staining. For lipid extraction, seed the cells onto a 10-cm cell culture dish10.</li>
	</ol>
	</li>
	<li>Cell treatment and Oil Red O staining
	<ol>
		<li>Replace the medium with 2 mL of starvation medium and incubate at 37 &#176;C for 24 h. Treat it with 30 mM glucose and 100 nM insulin. Incubate it in a 37 &#176;C culture incubator for 24 h.</li>
		<li>Aspirate the medium. Add 2 mL of PBS and swirl gently to rinse the medium; repeat once.</li>
		<li>Fix the coverslip to the slide with a rubber band. Expose the cells adherent to the outside surface. Perform the Oil Red O staining as described above (step 1.5). 
		NOTE: The basal levels of lipid accumulation are stimulated with a high glucose plus insulin treatment after serum starvation. The lipid drops are visualized by Oil Red O staining.</li>
	</ol>
	</li>
	<li>Measurement of lipids
	<ol>
		<li>Treat the hepatocytes with high glucose plus insulin in the same condition (step 2.2.1). Add 1 mL of PBS to each 10-cm cell culture dish. Scrape off the cells and harvest them into new 15-mL glass tubes, from which aliquots of 100 &#181;L are transferred to fresh 1.5-mL tubes for protein quantification.</li>
		<li>Add 4 mL of Chloroform/Methanol (2:1, v/v) to the remaining 900 &#181;L of cells. Sonicate for approximate 20 beeps and then vortex vigorously to release the lipid in the cells. Incubate the mixture on ice for 2 h. Perform the lipid extraction (step 1.6).</li>
	</ol>
	</li>
</ol>

<ol><li>Angulo, P. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/11961152">Medical progress - Nonalcoholic fatty liver disease.</a> New England Journal of Medicine. 346 (16), 1221-1231 (2002).</li>
<li>Chen, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hepatic+ATF6+Increases+Fatty+Acid+Oxidation+to+Attenuate+Hepatic+Steatosis+in+Mice+through+Peroxisome+Proliferator-Activated+Receptor+Alpha%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Hepatic ATF6 Increases Fatty Acid Oxidation to Attenuate Hepatic Steatosis in Mice through Peroxisome Proliferator-Activated Receptor Alpha.</a> Diabetes. 65 (7), 1904-1915 (2016).</li>
<li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((AMPK+phosphorylates+and+inhibits+SREBP+activity+to+attenuate+hepatic+steatosis+and+atherosclerosis+in+diet-induced+insulin-resistant+mice%5BTitle%5D)+AND+%22Cell+Metab%22%5BJournal%5D)">AMPK phosphorylates and inhibits SREBP activity to attenuate hepatic steatosis and atherosclerosis in diet-induced insulin-resistant mice.</a> Cell Metab. 13 (4), 376-388 (2011).</li>
<li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hepatic+SIRT1+attenuates+hepatic+steatosis+and+controls+energy+balance+in+mice+by+inducing+fibroblast+growth+factor+21%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">Hepatic SIRT1 attenuates hepatic steatosis and controls energy balance in mice by inducing fibroblast growth factor 21.</a> Gastroenterology. 146 (2), 539-549 (2014).</li>
<li>Esau, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((miR-122+regulation+of+lipid+metabolism+revealed+by+in+vivo+antisense+targeting%5BTitle%5D)+AND+%22Cell+Metabolism%22%5BJournal%5D)">miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting.</a> Cell Metabolism. 3 (2), 87-98 (2006).</li>
<li>Kanda, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MCP-1+contributes+to+macrophage+infiltration+into+adipose+tissue%2C+insulin+resistance%2C+and+hepatic+steatosis+in+obesity%5BTitle%5D)+AND+%22Journal+of+Clinical+Investigation%22%5BJournal%5D)">MCP-1 contributes to macrophage infiltration into adipose tissue, insulin resistance, and hepatic steatosis in obesity.</a> Journal of Clinical Investigation. 116 (6), 1494-1505 (2006).</li>
<li>Cohen, J. C., Horton, J. D., Hobbs, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+fatty+liver+disease%3A+old+questions+and+new+insights%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Human fatty liver disease: old questions and new insights.</a> Science. 332 (6037), 1519-1523 (2011).</li>
<li>Hebbard, L., George, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+models+of+nonalcoholic+fatty+liver+disease%5BTitle%5D)+AND+%22Nat+Rev+Gastroenterol+Hepatol%22%5BJournal%5D)">Animal models of nonalcoholic fatty liver disease.</a> Nat Rev Gastroenterol Hepatol. 8 (1), 35-44 (2011).</li>
<li>Chen, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Highly+effective+inhibition+of+lung+cancer+growth+and+metastasis+by+systemic+delivery+of+siRNA+via+multimodal+mesoporous+silica-based+nanocarrier%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Highly effective inhibition of lung cancer growth and metastasis by systemic delivery of siRNA via multimodal mesoporous silica-based nanocarrier.</a> Biomaterials. 35 (38), 10058-10069 (2014).</li>
<li>Gong, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fibroblast+growth+factor+21+improves+hepatic+insulin+sensitivity+by+inhibiting+mammalian+target+of+rapamycin+complex+1+in+mice%5BTitle%5D)+AND+%22Hepatology%22%5BJournal%5D)">Fibroblast growth factor 21 improves hepatic insulin sensitivity by inhibiting mammalian target of rapamycin complex 1 in mice.</a> Hepatology. 64 (2), 425-438 (2016).</li>
<li>Anstee, Q. M., Targher, G., Day, C. P. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/23507799">Progression of NAFLD to diabetes mellitus, cardiovascular disease or cirrhosis.</a> Nat Rev Gastroenterol Hepatol. 10 (6), 330-344 (2013).</li>
</ol>

The authors declare that they have no competing financial interests.
Funding: This work was supported by grants from the National Natural Science Foundation of China (No. 81270930, 31471129, and 31671224) and the Hundred Talents Program of the Chinese Academy of Sciences (2013OHTP04) to Y.L.

NAFLD is a series of progressive liver diseases that is associated with metabolic syndrome, obesity, insulin resistance, or type 2 diabetes mellitus (T2DM)11. The hallmark of NAFLD is steatosis, the accumulation of lipid in hepatocytes. Here, a spectrum of methods is presented to characterize the phenotypes and parameters of hepatic steatosis using DIO mice and mouse primary hepatocytes. This procedure could be helpful to elucidate the molecular mechanisms of NAFLD and other related metabolic dise...

Obesity is a burgeoning health problem in developed and developing countries. It has been reported to be one of the coexisting conditions frequently associated with nonalcoholic fatty liver disease (NAFLD), with a prevalence ranging between 30 and 100 percent in NAFLD patients1. Due to the strong correlation between fatty liver and obesity, diet-induced obese (DIO) mouse models are widely used to study the complex molecular mechanisms associated with the development of NAFLD2,3,4,5,6. Hepatic steatosis is the earliest stage of NAFLD, and it can progress to nonalcoholic steatohepatitis (NASH), cirrhosis, and ultimately, liver cancer7. Therefore, the overall goal of this method is to generate animal and cell models of hepatic steatotic conditions and to provide detailed protocols for efficient and accurate lipid measurement. These models and measurements are also useful for the investigation of other metabolic disorders, such as insulin resistance and type 2 diabetes.
As obesity is identified to be one of the key risk factors for NAFLD, a high-fat, high-sucrose diet (HFHS) that imitates the Western-style high-fat diet is used to induce obesity in mice. Subsequently, the degree of hepatic steatosis can be assessed using different methods. First, body weight and body composition analysis with nuclear magnetic resonance (NMR) show the lipid accumulation during feeding time. The fat mass and lean mass can be quantified in a non-invasive and real-time manner.
In addition, magnetic resonance imaging (MRI) is used to show both the whole-body and the liver distribution of fat. The grayscale signal of the MRI analysis can be converted into a legible pseudo-color image, and the intensity of the grayscale and color is hemi-quantifiable. This technology provides unique advantages for the measurement of lipid accumulation in live animals. Second, histological analysis of the liver is the most commonly used method to determine hepatic steatosis. Hematoxylin and Eosin (H&#38;E) staining provides histological information, such as hepatocyte morphology and macrophage infiltration, while Oil Red O staining shows the size and position of the lipid droplets in hepatocytes. Third, the lipid content analysis using chloroform/methanol extraction is an accurate and quantitative measurement of hepatic lipids. Total triglyceride and cholesterol levels can be measured with biochemical methods. Importantly, lipid extraction analysis and Oil Red O staining can also be used in genetically manipulated or pharmaceutically treated hepatocytes.
The advantage of the present method is its utilization of multiple optimized approaches to generate hepatic steatotic models and to comprehensively characterize the phenotypes both in vivo and in vitro. The DIO mouse models can recapitulate the pathology and metabolic phenotypes of human fatty liver disease. Other metabolic parameters in humans can be replicated in this model as well8. The generation of the steatotic hepatocyte model in response to high glucose plus insulin is efficient, useful, and overcomes the limitation of costly and time consuming mouse work. Taken together, these methods are sufficient and essential for the study of hepatic lipid dysfunction and insulin resistance during nutrient overloading.

<table><tbody><tr><td>O.C.T compound</td><td>SAKURA</td><td>4583</td><td></td></tr><tr><td>Oil Red O</td><td>Sigma</td><td>O0625-25G</td><td></td></tr><tr><td>Infinity Triglycerides kit</td><td>Fisher&amp;nbsp;Scientific</td><td>TR22421</td><td></td></tr><tr><td>Infinity Cholesterol kit</td><td>Fisher&amp;nbsp;Scientific</td><td>TR13421</td><td></td></tr><tr><td>Collagen type I, Rat tail</td><td>Millipore</td><td>08-115</td><td></td></tr><tr><td>DMEM (low glucose)</td><td>Invitrogen</td><td>11885-092</td><td></td></tr><tr><td>Penicillin / Streptomycin</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>FBS</td><td>Invitrogen</td><td>10099141</td><td></td></tr><tr><td>PBS</td><td>cellgro</td><td>R21-040-CVR</td><td></td></tr><tr><td>HBSS</td><td>cellgro</td><td>20-021-CV</td><td></td></tr><tr><td>Insulin</td><td>TOCRIS Bioscience</td><td>3435</td><td>dissolve in PBS, 1 mM for stock</td></tr><tr><td>Glucose</td><td>Sigma</td><td>G8270-100G</td><td></td></tr><tr><td>Microscope</td><td>Olympus</td><td>BX53</td><td></td></tr><tr><td>Peristaltic pump</td><td>Longerpump</td><td>BT100-2J</td><td></td></tr><tr><td>10 cm cell culture dish</td><td>Corning</td><td>420167</td><td></td></tr><tr><td>6-well-plate</td><td>Corning</td><td>3516</td><td></td></tr><tr><td>BCA assay</td><td>Beyotime</td><td>P0010</td><td></td></tr><tr><td>Nuclear Magnetic Resonance</td><td>Niumag technology</td><td>MiniQMR23-060H-I</td><td></td></tr><tr><td>High fat high surcose diet (HFHS)</td><td>Research Diets</td><td>D12327</td><td></td></tr></tbody></table>

optimized analysis of in vivo and in vitro hepatic steatosis

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.

As shown in Figure 1A, the mouse body weight was increased to 45±1.2 g after 16 weeks of HFHS feeding, which is approximately 1.5-fold higher than chow diet feeding group. The NMR body composition analyses showing the fat mass and lean mass of mice are indicated (Figure 1B). The fat distributions of the whole body and of the liver were determined by MRI, and representative pseudo-color images in live, conscious mice are shown in Figure 1C-D</stron...

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 ...

The overall goal of this procedure is to provide optimized method to generate and characterize Hepatic Steatosis using In Vivo and In Vitro approaches. This method can help answer key questions in the Mechanistic Study of Hepatic de-replication of lipid and glucose metabolism during nutrient overloading and obesity. One advantage to this techniques is that the physiological progress of Hepatic Steatosis can be assessed noninvasively prior to histological and biochemical analysis.This method can provide insight into Fatty Liver Disease. It can also be applied to other metabolic diseases such as Insulin Resistance, Type II Diabetes, and obesity. Demonstrating the procedure will be Zhimin, Yamei, and Aoyuan.They are all graduate students who work on transcriptional regulation of lipid and glucose metabolism from my laboratory. After anesthetizing the donor mouse, take a blood sample by cardiac puncture and collect the blood in an EDTA line tube. To remove the liver, cut the diaphragm along the liver's length and carefully remove it from the entero colia.From the liver, prepare two tissue blocks for H and E staining and Oil Red O staining. Keep the remaining tissue to measure lipid content for which at least 20 milligrams is needed. After making a 0.5%stock solution from Oil Red O from powder, dilute it to a 0.3%solution in water and filter the dilution through a 0.22 micron filter.Then, let the solution settle for ten minutes at room temperature. Next, embed the liver in OCT compound in a plastic embedding box. Then, freeze it at negative 20 degrees Celsius for 30 minutes.Once frozen, make eight micron slices with a cryostat. Mount the slices to slides and incubate the slides at room temperature for 30 minutes. Now stain the slices.Inside a staining tub, moisten a paper towel with 10%formalin. Keep the slides in the tub for five minutes of vapor fixation at four degrees Celsius. Next, dip the slides into 60%isopropanol for three seconds, do this twice.Now, add a few drops of Oil Red O working solution to each slide. Cover all of the tissue. Then, incubate the slide at room temperature for 15 minutes.Keep the slides shielded from light as much as possible from this point forward. After the incubation, briefly rinse the slides in fresh 60%isopropanol two times. Then, gently rinse the slides twice with distilled water.After the rinses, remove as much water as possible from the slides without drying out the tissue. Then, immerse the slides in a bath of hematoxylin stain for one minute. To removed the hematoxylin, rinse the slides with running tap water for five to ten minutes.Then, gently rinse the slides with distilled water two times and store them in a fresh bath of distilled water until they are mounted. To mount the slides, melt glycerol jelly in a microwave. Then, place several drops of liquified jelly onto the tissue slices.Before the jelly cools, carefully apply cover slips without trapping air bubbles. Begin with isolated primary hepatocytes growing on collagen coated glass cover slips in a six-well plate. For this model, first change the medium to two milliliters of starvation medium and incubate the cells for 24 hours.After 24 hours of starvation, adjust the medium to contain 30 millimolar glucose and 100 nanomolar insulin and let the cells incubate for another 24 hours. The next day, remove the medium and wash the cells using two milliliters of PBS. Perform two washes.To analyze the cells, fix each cover slip to a slide with a rubber band with the cells on the outside surface. Then, use the described Oil Red O staining method. This procedure can be applied to cells other than hepatocytes.To begin, load 20 to 40 milligrams of liver tissue into a two milliliter plastic centrifuge tube. Then, add one milliliter of PBS and homogenize the tissue. Once homogenized, transfer the lysate to a new 15 milliliter glass tube.Next, add five milliliters of two parts chloroform to one part methanol and vortex the mixture vigorously for one minute. Then, let the mixture incubate for two hours buried in ice. After the incubation, separate the solutions by spinning the tube at 1, 650 G for ten minutes at four degrees Celsius.The bottom face contains chloroform and lipids. Collect and transfer the bulk of it to a new glass tube kept on ice. Then, continue the extraction.To the remaining phases, add 600 microliters of four millimolar magnesium chloride and 1.5 milliliters of chloroform. Vortex this mixture vigorously and incubate the emulsion on ice. After half an hour on ice, repeat the centrifugation step and collect the bulk of the bottom phase and then combine this with the first bottom phase collection.Discard the remaining layers. To purify the lipids, evaporate the chloroform under nitrogen gas in a 37 degrees Celsius bath. Then, thoroughly rinse down the sides of the tube using 200 microliters of 1%Triton X-100 and chloroform and repeat the chloroform evaporation step.Now, dissolve the lipids in 200 microliters of double distilled water and vortex the lipids into an emulsion. Then, using commercial kits, measure the total triglyceride and cholesterol levels in the sample. By feeding mice a high fat high sucrose diet, their weight increased by 50%after 16 weeks.The diet resulted in an obvious gross morphological change in the livers. H and E and Oil Red O staining revealed that over the 16 week feeding regimen, lipid droplets grew larger and occupied regions usually occupied by hepatocytes. Analysis of the fat composition, performed using the described extraction method, showed that the excess nutrient overload caused abnormal cholesterol and triglyceride accumulation in the liver.In a different experiment, primary hepatocytes were cultured and subjected to 24 hours of starvation followed by glucose and insulin treatment. After the treatment, Oil Red O staining showed accumulation of lipid droplets in the cells. Quantitative lipid analysis showed that triglyceride and cholesterol levels were high in the treated cells.After watching this video, you should have a good understanding of how to generate In Vivo and In Vitro Hepatic Steatosis models. Two studies in mice presents under nized Fatty Liver Disease, insulin resistance, and Type II Diabetes.

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Research

JoVE Journal

Medicine

In this video, we demonstrate in vitro staining of neutral lipids in hepatocytes to detect hepatic steatosis or excessive fat accumulation in liver. Excessive fat is accumulated in the cells in form of lipid droplets. Oil Red O is a lipid soluble dye that binds to intracellular neutral lipids in these lipid droplets. When observed under a light microscope, these intracellular lipid droplets appear intensely red in color.

Oil Red O Staining: A Technique for Staining Neutral Lipids in Hepatocytes for Detecting Hepatic Steatosis In Vitro

De novo lipogenesis and β-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 β-fatty acid oxidation and lipogenesis.

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

The present report describes an in vitro enzymatic assay to measure phosphatidylethanolamine methyltransferase activity using Leishmania cell extracts. This assay is based on the transfer of a radioactive methyl group from S-[Methyl-3H]adenosyl-L-methionine onto endogenous phosphatidylethanolamine.

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

In this study, we describe a detailed protocol for inducing liver vesicular steatosis in differentiated HepaRG cells with the fatty acid salt sodium oleate and employ methods for detection and quantification of lipid accumulation, including coherent anti-Stokes Raman scattering (CARS) microscopy, cytofluorimetric analysis, Oil red O staining, and...

Inducing and Characterizing Vesicular Steatosis in Differentiated HepaRG Cells

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.

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

Fatty acid β-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 β-oxidation in freshly isolated primary hepatocytes using 14C-labeled palmitic acid.

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

Using a diet-induced non-alcoholic fatty liver disease (NAFLD) mouse model, we describe the use of novel in vivo micro-computed tomography imaging techniques as a non-invasive method to assess the progression stages of NAFLD, focusing predominantly on the hepatic vascular network due to its significant involvement in NAFLD-related hepatic dysregulation.

Novel In Vivo Micro-Computed Tomography Imaging Techniques for Assessing the Progression of Non-Alcoholic Fatty Liver Disease

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.

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

This article describes the use of oleic acid-induced HepG2 cells as a model for metabolic dysfunction-associated steatotic liver disease.

In Vitro Modeling of Fat Deposition in Metabolic Dysfunction-Associated Steatotic Liver Disease

An optical system was developed to visualize hepatic microcirculation with FITC-labeled erythrocytes and to measure the partial pressure of oxygen in the microvessels with laser-assisted phosphorimetry. This method can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, diameter, blood flow velocity, and oxygen tension.

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

Learn optimized methods to generate and characterize hepatic steatosis using in vivo and in vitro approaches. These techniques enable noninvasive assessment of liver lipid metabolism and disease progression.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

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