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 border="0" cellpadding="0" cellspacing="0" width="728">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">O.C.T compound</td>
			<td style="width:183px;">SAKURA</td>
			<td style="width:163px;">4583</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Oil Red O</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:163px;">O0625-25G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Infinity Triglycerides kit</td>
			<td style="width:183px;">Fisher&#160;Scientific</td>
			<td style="width:163px;">TR22421</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Infinity Cholesterol kit</td>
			<td style="width:183px;">Fisher&#160;Scientific</td>
			<td style="width:163px;">TR13421</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Collagen type I, Rat tail</td>
			<td style="width:183px;">Millipore</td>
			<td style="width:163px;">08-115</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">DMEM (low glucose)</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:163px;">11885-092</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Penicillin / Streptomycin</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:163px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">FBS</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:163px;">10099141</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">PBS</td>
			<td style="width:183px;">cellgro</td>
			<td style="width:163px;">R21-040-CVR</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">HBSS</td>
			<td style="width:183px;">cellgro</td>
			<td style="width:163px;">20-021-CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Insulin</td>
			<td style="width:183px;">TOCRIS Bioscience</td>
			<td style="width:163px;">3435</td>
			<td>dissolve in PBS, 1mM for stock</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Glucose</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:163px;">G8270-100G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Microscope</td>
			<td style="width:183px;">Olympus</td>
			<td style="width:163px;">BX53</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Peristaltic pump</td>
			<td style="width:183px;">Longerpump</td>
			<td style="width:163px;">BT100-2J</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">10cm cell culture dish</td>
			<td style="width:183px;">Corning</td>
			<td style="width:163px;">420167</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">6-well-plate</td>
			<td style="width:183px;">Corning</td>
			<td style="width:163px;">3516</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">BCA assay</td>
			<td style="width:183px;">Beyotime</td>
			<td style="width:163px;">P0010</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Nuclear Magnetic Resonance</td>
			<td style="width:183px;">Niumag technology</td>
			<td style="width:163px;">MiniQMR23-060H-I</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">High fat high surcose diet(HFHS)</td>
			<td style="width:183px;">Research Diets</td>
			<td style="width:163px;">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</...

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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 ...

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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. ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Monitoring of Systemic and Hepatic Hemodynamic Parameters in Mice

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. However, due to the small size of the mouse, technical details of the intraoperative monitoring procedure suitable for the mouse were rarely described. Previously we have reported a monitoring procedure to obtain hemodynamic parameters for rats. Now, we adapted the procedure to acquire systemic and hepatic hemodynamic parameters in mice, a species ten-fold smaller than rats. This film demonstrates the instrumentation of the animals as well as the data acquisition process needed to assess systemic and hepatic hemodynamics in mice. Vital parameters, including body temperature, respiratory&#160;rate&#160;and heart&#160;rate were recorded throughout the whole procedure. Systemic hemodynamic parameters consist of carotid artery pressure (CAP) and central venous pressure (CVP). Hepatic perfusion parameters include portal vein pressure (PVP), portal flow rate as well as the flow rate of the common hepatic artery (table 1). Instrumentation and data acquisition to record the normal values was completed within 1.5 h. Systemic and hepatic hemodynamic parameters remained within normal ranges during this procedure.
This procedure is challenging but feasible. We have already applied this procedure to assess hepatic hemodynamics in normal mice as well as during 70% partial hepatectomy and in liver lobe clamping experiments. Mean PVP after resection (n= 20), was 11.41&#177;2.94 cmH2O which was significantly higher (P&#60;0.05) than before resection (6.87&#177;2.39 cmH2O). The results of liver lobe clamping experiment indicated that this monitoring procedure is sensitive and suitable for detecting small changes in portal pressure and portal flow rate. In conclusion, this procedure is reliable in the hands of an experienced micro-surgeon but should be limited to experiments where mice are absolutely needed.

This film demonstrates how to acquire systemic and hepatic hemodynamics in mice. The whole monitoring includes acquisition of vital parameters, systemic blood pressure, central venous pressure, common hepatic artery flow rate, and portal vein pressure as well as the portal flow rate in mice.

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. ...

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Preparation Of Gushukang (GSK) Granules for In Vivo and In Vitro Experiments

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang (GSK) granules, a marketed prescription in China, have bone-protective effects in treating POP. Before administration to the body, one standard preparation procedure is commonly required, which aims to promote the release of active constituents from raw herbs and enhance the pharmacological effects as well as therapeutic outcomes. This study proposes a detailed protocol for using GSK granules in in vivo and in vitro experimental assays. The authors first provide a detailed protocol to calculate the animal-appropriate dosages of granules for in vivo investigation: weighing, dissolving, storage, and administration. Second, this article describes protocols for micro-CT scanning and the measurement of bone parameters. Sample preparation, protocols for running the micro-CT machine and quantification of bone parameters were evaluated. Third, serum-containing GSK granules are prepared, and drug-containing serum is extracted for in vitro osteoclastogenesis and osteoblastogenesis. GSK granules were intragastrically administered twice per day to rats for three consecutive days. Blood was then collected, centrifuged, inactivated, and filtered. Finally, serum was diluted and used for performing osteoclastogenesis and osteoblastogenesis. The protocol described here can be considered a reference for pharmacological investigations of herbal prescription medicines, such as granules.

Preparation Of Gushukang GSK Granules for In Vivo and In Vitro Experiments

This article provides a detailed protocol for preparing a working solution of Gushukang granules for animal studies and GSK granule containing serum for in vitro experiments. This protocol can be applied to pharmacological investigations of herbal medicines as well as prescriptions for both in vivo and in vitro experiments.

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang ...

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the &#946;3 subunit of voltage-gated calcium channels (Cav&#946;3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cav&#946;3-deficient mice (Cav&#946;3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cav&#946;3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cav&#946;3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cav&#946;3-deficient mice or cells might also be applicable for specific molecules other than Cav&#946;3.

Here, we present a protocol for an in vitro scratch assay using primary fibroblasts and for an in vivo skin wound healing assay in mice. Both assays are straightforward methods to assess in vitro and in vivo wound healing.

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective ...