Name: optimized analysis of in vivo and in vitro hepatic steatosis
Uploaded: 2017-03-11T15:00:00.000+00:00
Duration: 8 min 58 s
Description: Watch this Scientific Journal Video about Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis at JoVE.com

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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medical+progress+-+Nonalcoholic+fatty+liver+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+ATF6+Increases+Fatty+Acid+Oxidation+to+Attenuate+Hepatic+Steatosis+in+Mice+through+Peroxisome+Proliferator-Activated+Receptor+Alpha.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPK+phosphorylates+and+inhibits+SREBP+activity+to+attenuate+hepatic+steatosis+and+atherosclerosis+in+diet-induced+insulin-resistant+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+SIRT1+attenuates+hepatic+steatosis+and+controls+energy+balance+in+mice+by+inducing+fibroblast+growth+factor+21.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-122+regulation+of+lipid+metabolism+revealed+by+in+vivo+antisense+targeting.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MCP-1+contributes+to+macrophage+infiltration+into+adipose+tissue,+insulin+resistance,+and+hepatic+steatosis+in+obesity.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+fatty+liver+disease:+old+questions+and+new+insights.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+nonalcoholic+fatty+liver+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+effective+inhibition+of+lung+cancer+growth+and+metastasis+by+systemic+delivery+of+siRNA+via+multimodal+mesoporous+silica-based+nanocarrier.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+factor+21+improves+hepatic+insulin+sensitivity+by+inhibiting+mammalian+target+of+rapamycin+complex+1+in+mice.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+of+NAFLD+to+diabetes+mellitus,+cardiovascular+disease+or+cirrhosis.">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

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&#177;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</...

Watch this Scientific Journal Video about Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis at JoVE.com

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.

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 ...

optimized-analysis-of-in-vivo-and-in-vitro-hepatic-steatosis

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

15.8K Views.  Chinese Academy of Sciences. 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. 

Video: Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

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

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of the hepatic blood supply is poorly oxygenated portal vein blood derived from the gastrointestinal tract and spleen. Oxygen is delivered to hepatocytes by blood flowing from a terminal branch of the portal vein to a central venule via sinusoids, and this makes an oxygen gradient in hepatic lobules. The oxygen gradient is an important physical parameter that involves the expression of enzymes upstream and downstream in hepatic microcirculation, but the lack of techniques for measuring oxygen consumption in the hepatic microcirculation has delayed the elucidation of mechanisms relating to oxygen metabolism in liver. We therefore used FITC-labeled erythrocytes to visualize the hepatic microcirculation and used laser-assisted phosphorimetry to measure the partial pressure of oxygen in the microvessels there. Noncontact and continuous optical measurement can quantify blood flow velocities, vessel diameters, and oxygen gradients related to oxygen consumption in the liver. In an acute hepatitis model we made by administering acetaminophen to mice we observed increased oxygen pressure in both portal and central venules but a decreased oxygen gradient in the sinusoids, indicating that hepatocyte necrosis in the pericentral zone could shift the oxygen pressure up and affect enzyme expression in the periportal zone. In conclusion, our optical methods for measuring hepatic hemodynamics and oxygen consumption can reveal mechanisms related to hepatic 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.

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

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 ...

Inducing and Characterizing Vesicular Steatosis in Differentiated HepaRG Cells

Hepatic steatosis represents a metabolic dysfunction that results from an accumulation of triglyceride-containing lipid droplets in hepatocytes. Excessive fat accumulation leads to non-alcoholic fatty liver disease (NAFLD), &#160;which is potentially reversible and may evolve into non-alcoholic steatohepatitis (NASH) and eventually cirrhosis and hepatocellular carcinoma (HCC). The molecular mechanisms linking lipid accumulation in hepatocytes with the progression to NASH, irreversible liver damage, fibrosis, cirrhosis, and even HCC still remains unclear. To this end, several in vitro and in vivo models have been developed to elucidate the pathological processes that cause NAFLD. In the present study, we describe a cellular model for the induction of liver vesicular steatosis that consists of DMSO-differentiated human hepatic HepaRG cells treated with the fatty acid salt sodium oleate. Indeed, sodium oleate-treated HepaRG cells accumulate lipid droplets in the cytoplasm and show typical features of steatosis. This in vitro human model represents a valuable alternative to in vivo mice models as well as to the primary human hepatocytes. We also present a comparison of several methods for the quantification and evaluation of fat accumulation in HepaRG cells, including Oil Red O staining, cytofluorimetric Bodipy measurement, metabolic gene expression analysis by qPCR, and coherent anti-Stokes Raman scattering (CARS) microscopy. CARS imaging combines the chemical specificity of Raman spectroscopy, a chemical analysis technique well-known in materials science applications, with the benefits of high-speed, high-resolution non-linear optical microscopies to allow precise quantification of lipid accumulation and lipid droplet dynamics. The establishment of an efficient in vitro model for the induction of vesicular steatosis, alongside an accurate method for the quantification and characterization of lipid accumulation, could lead to the development of early stage diagnosis of NAFLD via the identification of molecular markers, and to the generation of new treatment strategies.

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 qPCR.&#160;

Hepatic steatosis represents a metabolic dysfunction that results from an accumulation of triglyceride-containing lipid droplets in hepatocytes. Excessive ...

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 ...

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

Non-alcoholic fatty liver disease (NAFLD) is a growing global health issue, and the impact of NAFLD is compounded by the current lack of effective treatments. Considerable limiting factors hindering the timely and accurate diagnosis (including grading) and monitoring of NAFLD, as well as the development of potential therapies, are the current inadequacies in the characterization of the hepatic microenvironment structure and the scoring of the disease stage in a spatiotemporal and non-invasive manner. Using a diet-induced NAFLD mouse model, we investigated the use of in vivo micro-computed tomography (CT) 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. This imaging methodology allows for longitudinal analysis of liver steatosis and functional tissue uptake, as well as the evaluation of the relative blood volume, portal vein diameter, and density of the vascular network. Understanding the adaptations of the hepatic vascular network during NAFLD progression and correlating this with other ways of characterizing the disease progression (steatosis, inflammation, fibrosis) using the proposed method can pave the way toward the establishment of new, more efficient, and reproducible approaches for NAFLD research in mice. This protocol is also expected to upgrade the value of preclinical animal models for investigating&#160;the development of novel therapies against disease progression.

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

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.

Non-alcoholic fatty liver disease (NAFLD) is a growing global health issue, and the impact of NAFLD is compounded by the current lack of effective ...

Oleic Acid-Induced HepG2 Cells for Metabolic Disease Analysis 

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

The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) has surged due to changes in economic and lifestyle patterns, leading to significant health challenges. Previous reports have studied the establishment of animal and cellular models for MASLD, highlighting differences between them. In this study, a cellular model was created by inducing fat accumulation in MASLD. HepG2 cells were stimulated with the unsaturated fatty acid oleic acid at various concentrations (0.125 mM, 0.25 mM, 0.5 mM, 1 mM) to emulate MASLD. The model&#39;s efficacy was assessed using cell counting kit-8 assays, Oil Red O staining, and lipid content analysis. This study aimed to create a simple-to-operate cellular model for MASLD cells. Results from the cell counting kit-8 assays showed that the survival of HepG2 cells was dependent on the concentration of oleic acid, with a GI50 of 1.875 mM. Cell viability in the 0.5 mM and 1 mM groups were significantly lower than those in the control group (P &#60; 0.05). Furthermore, Oil Red O staining and lipid content analysis examined fat deposition at varying oleic acid concentrations (0.125 mM, 0.25 mM, 0.5 mM, 1 mM) on HepG2 cells. The lipid content of the 0.25 mM, 0.5 mM, and 1 mM groups was significantly higher than that of the control group (P &#60; 0.05). Additionally, triglyceride levels in the OA groups were significantly higher than those in the control group (P &#60; 0.05).

Author Spotlight: Establishing MASLD Cell Models for Investigating Disease Mechanisms and the Lipid-Lowering Effects of Koumiss

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

The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) has surged due to changes in economic and lifestyle patterns, leading ...

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

Put a cell culture coverslip in every well in a 24-well plate, and seed HepG2 cells, as described in the text manuscript. After the appropriate incubation, wash the cells with 1 milliliter of PBS, and discard the supernatant. Fix them with 1 milliliter of 4% paraformaldehyde in PBS, and incubate for 1 hour at room temperature. Discard the excess paraformaldehyde, and rinse the cells with 1 milliliter of distilled water.
Then, add 1 milliliter of 70% isopropanol, and incubate for 5 minutes. Discard the excess isopropanol, and add 1 milliliter of Oil-red O solution, and incubate for 30 minutes. Discard the excess Oil-red O solution, and then, rinse with 1 milliliter of distilled water. Observe the cells under the microscope at a magnification of 400x.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

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Inducing and Characterizing Vesicular Steatosis in Differentiated HepaRG Cells

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

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

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

Barwienie czerwienią olejową O: technika barwienia obojętnych lipidów w hepatocytach w celu wykrycia stłuszczenia wątroby in vitro