Name: isolation of primary mouse hepatocytes for nascent protein synthesis analysis by non-radioactive l-azidohomoalanine labeling method
Uploaded: 2018-10-23T13:30:00
Description: Watch this Scientific Journal Video about Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method at JoVE.com

We thank Drs. Joonbae Seo and Vivian Hwa for their scientific input and discussion. This work was supported by National Institute of Health (NIH) (R01DK107530). T.N. was supported by the PRESTO from the Japan Science and Technology Agency. A part of this study was supported by a grant from NIH (P30DK078392) for the Digestive Disease Research Core Center in Cincinnati.

This protocol contains the use of laboratory mice. Animal care and experimental procedures were performed according to procedures approved by the animal care committees of Cincinnati Children Hospital Medical Center.
1. Isolation of Primary Mouse Hepatocytes
<ol>
	<li>Pre-isolation preparations
	<ol>
		<li>Prepare 450 mL of 40% density gradient buffer as described in Table 1 and keep at 4 &#176;C (15 mL/mouse).</li>
		<li>Prepare 500 mL of Williams&#8217; Medium as described...

Although several immortalized hepatic cell lines have been proposed and used to investigate liver functions49,50,51,52, these cells generally lack the important and fundamental functions of normal hepatocytes, such as the expression of albumin (Figure 3). It is widely recognized, therefore, that utilizing primary hepatocytes is a valuable option for examining liver...

Protein is an important nutritional element and approximately 50% of the dry weight of a human body is composed of proteins which have several biological traits and functions1. Consequently, protein synthesis is one of the most energy consuming events and an alteration in protein metabolism is highly associated with the development of diseases, including metabolic diseases2,3,4. In the liver, protein biosynthesis accounts for approximately 20&#8211;30% of total energy consumption5,6. In addition, proteins function as not only passive or building blocks of the liver, but also active signal-mediating factors intracellularly or extracellularly to regulate the systemic metabolism7. For instance, reduced levels of serum albumin, which is synthesized and secreted by the liver and the most abundant protein in the plasma8, increases the risk of type 2 diabetes development9,10,11, whereas a higher concentration of serum albumin is protective against developing metabolic syndrome12. Furthermore, disturbed or disruption of secretory or membrane-bound hepatic proteins, which modulate cholesterol homeostasis, including lipoproteins, LDLR, and LRP1, can lead to the development of insulin resistance, hyperlipidemia, or atherosclerosis13. Therefore, the identification of molecular pathophysiological mechanisms that are involved in protein metabolism disturbance in the liver and its associated metabolic complications might be useful for discovering novel pharmacological approaches to retard onset or treat metabolic diseases such as insulin resistance, diabetes, and non-alcoholic fatty liver.
Protein synthesis is tightly linked to the cellular energy status (e.g., the formation of one peptide bond during the elongation step of protein synthesis requires 4 phosphodiester bonds14) and is regulated by molecular pathways that sense inter- and intra-cellular nutrient availability15,16. AMP-activated protein kinase (AMPK) is one of the intracellular energy sensors that maintain energy homeostasis17. Once AMPK is activated when cellular energy levels become lower, AMPK and its targeted substrates function to stimulate catabolic pathways and inhibit anabolic processes including protein synthesis18,19. The regulation of protein synthesis is mediated by phosphorylation of multiple translation factors and ribosomal proteins20. Of note, the mammalian target of rapamycin complex 1 (mTORC1), a major driver of protein synthesis, is one of the main targets of AMPK21. Activation of the mTORC1 pathway enhances the cell growth and proliferation by stimulating protein translation and autophagy20,21. Therefore, it is logical that activation of AMPK can inhibit mTORC1-mediated protein synthesis22. Indeed, activation of AMPK counteracts and directly phosphorylates mTORC1 on threonine residue 2446 (Thr2446) leading to its inactivation23 and suppression of protein biosynthesis24. Moreover, AMPK can indirectly inhibit mTORC1 function by phosphorylation and activation of tuberous sclerosis complex 2 (TSC2)25 which is the upstream regulator of mTORC1 signaling cascade. In short, dysregulation of these pathways in the liver is often linked to the development of metabolic diseases and therefore there is a critical need to establish effective experimental tools to investigate the role of these pathways in the regulation of energy and protein metabolism in hepatocytes.
There is a stronger similarity between the functional properties of isolated primary hepatocytes and in vivo hepatocytes than with in vitro liver-derived cell lines26,27,28. It has been shown that primary human hepatocytes share 77% similarity with that of liver biopsies, whereas HepG2 cells, which are well-differentiated hepatic cancerous cells and widely used to investigate hepatic functions, display less than 48% in the context of gene expression profiles29. Therefore, utilization of primary hepatocytes, rather than immortalized culture cells, is of vital importance in investigating hepatic function and physiology, and several protocols are available for the isolation and culture of primary hepatocytes especially from rats30,31. While the rat hepatocytes are useful with a relatively higher yield of cells, mouse hepatocytes have a greater potential in many scientific aspects because of the wide availability of genetically modulated mice. However, there are several technical challenges in isolating healthy and abundant primary hepatocytes from mice for cellular- and molecular-based assessments: first, cannula insertion to perfuse the liver with buffer reagents is very difficult to handle because of the small and thin mouse portal vein or inferior vena cava; second, a longer manipulation time of cells during the isolation can cause reduction in cell quantity and quality; third, non-enzymatic mechanical separation methods can result in severe damage and produce a low yield of viable isolated primary hepatocytes32,33. In the 1980s, collagenase perfusion technique was introduced for isolating hepatocytes from the livers of animals34. This method is based on collagenase perfusion of the liver35,36,37, infusion of the liver with calcium chelator solution38,39, enzymatic digestion and mechanical dissociation of the hepatic parenchyma35. In the first step, a mouse liver is perfused with a calcium [Ca2+] free buffer containing a [Ca2+] chelator (ethylenediamine tetraacetic acid, EDTA). In the second step, the mouse liver is perfused with a collagenase-containing buffer to hydrolyze the cellular-extracellular matrix interactions. Unlike the buffer used in step one, the presence of [Ca2+] ions in the buffer of the second step is required for effective collagenase activity, after which the digested liver has to be further gently and mechanically separated using forceps between the hepatic capsule and parenchymatous tissue. Finally, connective tissue is removed by filtering, and subsequent centrifugation separates viable hepatocytes from both non-parenchymal cells and non-living hepatocyte with the use of density gradient buffer40,41,42. In the present study, we show a modified two-step collagenase perfusion technique to isolate primary hepatocytes from a mouse liver for the analysis of protein synthesis.
The radiolabeling of proteins is widely used to quantify the expression levels, turnover rates, and determine the biological distribution of proteins43 due to the high sensitivity of detection of radioactivity44. However, the use of radioactive isotopes requires highly controlled research circumstances and procedures45. Alternative non-radioactive methods have been developed and have increasingly gained in popularity. The chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method is one of them and is based on the chemo-selective reaction between an azide and alkyne groups46, which can be utilized to analyze cellular events such as detecting nascent protein synthesis and subclasses of glycoproteins modified with an azide group. For nascent protein synthesis, L-Azidohomoalanine (L-AHA, an azide-modified amino acid) can be metabolically incorporated into proteins and detected by using the TAMRA protein detection method47. By using this assay in primary mouse hepatocytes, we show that the nascent protein synthesis rate is tightly linked to the availability of ATP from mitochondrial and AMPK activation (Figure 1).
In summary, utilization of primary mouse hepatocytes is crucial to investigating the protein and energy metabolism and quantifying nascent protein synthesis is valuable for gaining insights into the physiological role of pathways relevant to the development and cure of hepatocyte-related diseases.

<table>
	<tbody>
		<tr>
			<td>HEPES buffer</td>
			<td>Fisher Scientific</td>
			<td>BP310-500</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Fisher Scientific</td>
			<td>D16-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene glycol-bis(&#946;-aminoethyl ether)-tetraacetic acid</td>
			<td>AmericanBio</td>
			<td>AB00505-00025</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100X)</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (10X) no calcium, magnesium, phenol red</td>
			<td>Gibco</td>
			<td>14185-052</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride Dihydrate (CaCl2.2H2O)</td>
			<td>Fisher Scientific</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Density gradient buffer</td>
			<td>GE Healthcare</td>
			<td>17-0891-02</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (Dulbecco&#39;s Modified Eagle Medium) low glucose, pyruvate</td>
			<td>Gibco</td>
			<td>11885-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Hyclone</td>
			<td>SH30910.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS) (1X)</td>
			<td>Gibco</td>
			<td>1897141</td>
			<td></td>
		</tr>
		<tr>
			<td>Williams medium E, no glutamine</td>
			<td>Gibco</td>
			<td>12551-032</td>
			<td></td>
		</tr>
		<tr>
			<td>L-alanyl-L-glutamine dipeptide supplement</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type X</td>
			<td>Wako Pure Chemical Industries</td>
			<td>039-17864</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion pump</td>
			<td>Cole-Parmer</td>
			<td>Masterflex L/S</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>IV administration set</td>
			<td>EXELINT</td>
			<td>29081</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>A water bath</td>
			<td>REVSCI</td>
			<td>RS-PB-200</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Tube heater</td>
			<td>Fisher Scientific</td>
			<td>Isotemp</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon Lab, Inc</td>
			<td>0-39613</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>PHOENIX</td>
			<td>10250</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclaved Cotton Tips</td>
			<td>Fisherbrand</td>
			<td>23-400-124</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm Petri Dish</td>
			<td>TPP</td>
			<td>93100</td>
			<td></td>
		</tr>
		<tr>
			<td>Connector (Male Luer Lock Ring)</td>
			<td>Cole-Parmer instrument</td>
			<td>EW-4551807</td>
			<td></td>
		</tr>
		<tr>
			<td>24G catheters</td>
			<td>TERUMO</td>
			<td>Surflo 24Gx3/4&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#956;m Filter (CELL STRAINERS)</td>
			<td>VWR</td>
			<td>10199-658</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml conical-bottom centrifuge tubes</td>
			<td>VWR</td>
			<td>89039-666</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml conical-bottom centrifuge tubes</td>
			<td>VWR</td>
			<td>89039-658</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemoselective ligation reaction PROTEIN ANALYSIS DETECTION KIT, TAMRA ALKYNE</td>
			<td>Invitrogen</td>
			<td>C33370</td>
			<td></td>
		</tr>
		<tr>
			<td>AHA (L-azidohomoalanine)</td>
			<td>Invitrogen</td>
			<td>C10102</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (methionine free)</td>
			<td>Gibco</td>
			<td>21013024</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cystine Dihydrochloride</td>
			<td>SIGMA</td>
			<td>C2526</td>
			<td></td>
		</tr>
		<tr>
			<td>Laemmli sample buffer</td>
			<td>BioRad</td>
			<td>161-0737</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail</td>
			<td>SIGMA</td>
			<td>P9599</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS solution (20%)</td>
			<td>BioRad</td>
			<td>161-0418</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCL (1M)</td>
			<td>American Bioanalytical</td>
			<td>AB14044-01000</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphatase Inhibitor Cocktail</td>
			<td>SIGMA</td>
			<td>P5726</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein concentration measuring Kit (Bovin Serum Albumin-BSA)</td>
			<td>BioRad</td>
			<td>500-0207</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture plate</td>
			<td>TPP</td>
			<td>92006</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Heatblock</td>
			<td>VWR</td>
			<td>12621-092</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Multi-Rotator</td>
			<td>Grant-bio</td>
			<td>PTR-60</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Ultrasonic Sonicator</td>
			<td>Cole-Parmer</td>
			<td>GE130PB</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Standard Heavy-Duty Vortex Mixer</td>
			<td>VWR</td>
			<td>97043-566</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>A variable mode laser scanner</td>
			<td>GE Healthcare Life Science</td>
			<td>FLA 9500</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Coomassie-dye reagent</td>
			<td>Thermo Scientific</td>
			<td>24594</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Olympus</td>
			<td>CKX53</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Western Blotting apparatus</td>
			<td>BioRad</td>
			<td>1658004</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424R</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Automated cell counter</td>
			<td>BioRad</td>
			<td>TC20</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>FluorChem R system</td>
			<td>proteinsimple</td>
			<td>-</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>p-Ampka (T172) antibody</td>
			<td>Cell signaling</td>
			<td>2535</td>
			<td></td>
		</tr>
		<tr>
			<td>Total-AMPK antibody</td>
			<td>Cell signaling</td>
			<td>5832</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin antibody</td>
			<td>Cell signaling</td>
			<td>4929</td>
			<td></td>
		</tr>
		<tr>
			<td>beta actin antibody</td>
			<td>Santa Cruz</td>
			<td>sc-130656</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine scissors and forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of primary mouse hepatocytes for nascent protein synthesis analysis by non-radioactive l-azidohomoalanine labeling method

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for systemic energy homeostasis. Primary hepatocytes isolated from the murine liver constitute a valuable biological tool to understand the functional properties or alterations occurring in the liver. Herein we describe a method for the isolation and culture of primary mouse hepatocytes by performing a two-step collagenase perfusion technique and discuss their utilization for investigating protein metabolism. The liver of an adult mouse is sequentially perfused with ethylene glycol-bis tetraacetic acid (EGTA) and collagenase, followed by the isolation of hepatocytes with the density gradient buffer. These isolated hepatocytes are viable on culture plates and maintain the majority of endowed characteristics of hepatocytes. These hepatocytes can be used for assessments of protein metabolism including nascent protein synthesis with non-radioactive reagents. We show that the isolated hepatocytes are readily controlled and comprise a higher quality and volume stability of protein synthesis linked to energy metabolism by utilizing the chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method and western blotting analyses. Therefore, this method is valuable for investigating hepatic nascent protein synthesis linked to energy homeostasis. The following protocol outlines the materials and methods for the isolation of high-quality primary mouse hepatocytes and detection of nascent protein synthesis.

Primary mouse hepatocytes isolation results in a yield of approximately 20 x 106 total cells/mouse. Histologically, live and attached primary hepatocytes appear polygonal or typical hexagonal in shape with clearly outlined membranous boundary after 24 h incubation (Figure 2).
To confirm whether isolated cells are primary hepatocytes, we compared expression levels of albumin protein in iso...

Watch this Scientific Journal Video about Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method at JoVE.com

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

Here, we present a protocol for the isolation of healthy and functional primary mouse hepatocytes. Instructions for detecting hepatic nascent protein synthesis by non-radioactive labeling substrate were provided to help understand the mechanisms underlying protein synthesis in the context of energy-metabolism homeostasis in the liver.

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for ...

This method can help answer key questions in various metabolic disease research fields about molecular alterations of hepatic energy and protein biosynthesis in obesity, non-alcoholic fatty liver, and type two diabetes. The main advantages of this technique are that it facilitates the isolation of functional primary mouse hepatocytes and detection hepatic nascent proteins via a nonradioactive labeling substrate. Visual demonstration of this method is critical as the perfusion step is difficult to learn because of the small and thin mouse blood vessels.For liver perfusion, carefully insert a 24 gauge catheter into the Inferior Vena Cava or IVC just at the bifurcation with the right renal vein. Remove the catheter needle maintaining the position of the cannula within the IVC and connect a 24 gauge cannula with perfusion tube by using the connector. Begin perfusing the liver with warm HBSS minus at a four milliliter permanent flow rate quickly cutting the splenic vein to drain out the internal blood.After 10 minutes, perfuse the mouse liver with 35 milliliters of HBSS plus supplemented with Collagenase Type X at a flow rate of four milliliters per minute, clamping the splenic vein for about 10 seconds every few minutes to facilitate distribution of the perfusate throughout the liver. When all of the Collagenase has been perfused, transfer the liver onto a lab tissue before stopping the pump to avoid blood backflow into the liver. Use forceps to remove the gallbladder and gently wipe the liver with a fresh lab tissue to remove any blood.Place the liver in a 100 millimeter Petri dish containing five milliliters of fresh warm HBSS plus supplemented with Collagenase and use stray-tipped forceps to gently remove the liver capsule. Use the forceps to carefully disperse the parenchymal tissue and add 15 milliliters cold DMEM to the Petri dish. Shake the torn liver gently to release the residual parenchymal cells into the medium and add another 15 milliliters of cold DMEM to the dish to acquire the rest of the cells.Then filter the tissue slurry through a 100 micron cell strainer into a 50 milliliter conical tube on ice. To isolate the primary mouse hepatocytes, collect the cells by centrifugation and resuspend the pellet in 10 milliliters of fresh DMEM. Carefully layer the cells over a 40%density gradient buffer for density gradient separation, discarding the cells from the upper and middle layers after centrifugation.Resuspend the primary parenchymal hepatocyte pellet with two to three milliliters of William's Medium E to avoid collecting dead cells from the wall of the tube and transfer the cells into a new 50 milliliter tube. Mix the cells with an additional seven to eight milliliters of William's E Medium before centrifugation and resuspend the pellet in 10, 20, or 30 milliliters of fresh William's Medium E depending on the size of the pellet. After counting, seed six times 10 to the fifth cells to each well of a six-well plate for a two to three-hour incubation at 37 degrees Celsius.At the end of the incubation, replace the medium with DMEM supplemented with 10%Fetal Bovine Serum and anti-anti to remove the dead and unattached cells and return the cells to the incubator overnight. For azide-modified nascent protein labeling, add 20 to 40 micrograms of cell lysate to 50 microliters of 2X reaction buffer. Bring the volume to 80 microliters with distilled deionized water and vortex the protein solution for five seconds.Add five microliters of copper sulfate to the cells for another five-second vortex, followed by five microliters of reaction buffer additive one with five seconds of vortexing. After a three-minute incubation, add 10 microliters of reconstituted reaction buffer additive two to the cells for another five-second vortex and rotate the sample for 20 minutes at four degrees Celsius on a multi-rotator machine protected from light. At the end of the rotation, add 300 microliters of methanol to the lysate, followed by 75 microliters of chloroform, and 200 microliters of double distilled water.Collect the labeled protein by centrifugation and add 250 microliters of fresh methanol to the pellet. After vortexing, centrifuge the lysate again and cover the bare pellet with a lint-free tissue for 15 minutes at room temperature. For PAGE analysis, solubilize the dried pellet in 20 microliters of loading buffer without beta-mercaptoethanol and vortex the pellet for 10 minutes.Next, heat the protein in a 70 degree Celsius heat block for 10 minutes and load the sample onto a 10%sodium dodecyl sulfate gel for tetramethylrhodamine detection. Then use a variable mode laser scanner for the precise quantitation of the AHA-labeled nascent proteins. Histologically, live and attached primary hepatocytes appear typically polygonal or hexagonal in shape with a clearly outlined membranous boundary after 24 hours of culture.To confirm whether the isolated cells are primary hepatocytes, in this experiment, albumin protein expression levels were compared in mouse embryonic fibroblasts, a mouse hepatoma cell line, isolated primary mouse hepatocytes, and mouse livers. The albumin protein expression was detected in primary mouse hepatocytes and mouse livers, but was not detectable in either mouse embryonic fibroblasts or hepatoma cell line cells. Primary hepatocyte treatment with 10 micromolar Rotenone for four to six hours revealed a robust induction of hepatic AMP-activated protein kinase as detected by increased phosphorylation levels on hepatic AMP-activated protein kinase T172 similar to those observed in vivo.Indeed, five hours of 10 micromolar Rotenone treatment had profound inhibitory effects on nascent protein synthesis in treated primary hepatocytes compared to untreated control cells as demonstrated by a chemoselective ligation reaction assay. While attempting this procedure, it's important to remember to prepare all required reagents and mediums in advance. This technique paved the way for the researcher in the field of hepatic metabolic pathophysiology to explore the molecular mechanism of energy protein regulation in obesity and type two diabetes in isolated primary mouse hepatocyte.

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