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Method Article

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

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DOI:

10.3791/58323

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October 23rd, 2018

 ,  ,  ,  ,  ,  , 
1Department of Pharmacology and Systems Physiology, College of Medicine, University of Cincinnati, 2Division of Endocrinology, Cincinnati Children's Hospital Medical Center, 3Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, 4Department of Pediatrics, College of Medicine, University of Cincinnati

* These authors contributed equally

In This Article

Summary

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.

Abstract

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.

Introduction

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 20–30% 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.

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Protocol

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

  1. Pre-isolation preparations
    1. Prepare 450 mL of 40% density gradient buffer as described in Table 1 and keep at 4 °C (15 mL/mouse).
    2. Prepare 500 mL of Williams’ Medium as described in Table 1 and keep at 4 °C.
    3. Prepare 500 mL of DMEM as described in Table 1 and keep on ice (30 mL/mouse).
    4. Prepare 500 mL of HBSS (-) Buffer as described in Table 1 and keep at 42 °C in the water bath (40 mL/mouse).
    5. Prepare 500 mL of HBSS (+) Buffer with 0.3 mg/mL Collagenase Type X as described in Table 1 with 30 min incubation at 42 °C in the water bath (40 mL/mouse).
    6. Set up the perfusion pump by placing the intravenous (I.V) administration tubing across the latching foot pedal and by checking the flow of flushing solution without air bubbles.
    7. Warm up the tube heater at 42 °C.
    8. Cool down the centrifuge machine to 4 °C.
  2. Anesthesia and Perfusion
    1. Clean the work areas and the pump.
    2. Rinse out the pump-connected IV tube by running 70% ethanol thoroughly for 10 min.
    3. Remove the residual ethanol from the pump-connected IV tube by rinsing with DDW entirely for 10 min.
    4. Wipe the scissors and forceps with 70% ethanol.
    5. Place the mouse in a plastic container with a mesh bottom and with the 2 mL isoflurane-soaked gauze underneath the mesh to avoid the direct contact.
    6. Asses the depth of anesthesia by pedal reflex (firm toe pinch).
    7. Remove the mouse when it has reached the desired depth of anesthesia.
      NOTE: The induction time of anesthesia should not be more than 1 min.
    8. Construct a nose cone using a 15 mL conical tube with 1 mL isoflurane-soaked pad pushed to the end of the tube.
    9. Control the depth of anesthesia by moving the nose cone closer or away from the mouse nostrils.
    10. Place the mouse on the working platform with the ventral side facing up.
    11. Spray 70% ethanol over the abdominal area of the mouse and then open the abdominal cavity by making a large transverse incision of the dermis and peritoneum by using sharp scissors and toothed forceps.
    12. Then, make a vertical cut of the abdominal layers until all abdominal organs are completely exposed by using sharp scissors and toothed forceps.
    13. Move small and large intestines toward the left side of the mouse with autoclaved cotton tip until liver, portal vein, and inferior vena cava (IVC) are clearly exposed.
    14. Insert a 24 G catheter into IVC just at the bifurcation with right renal vein carefully without shaking hands to avoid the injury of IVC and bleeding.
      NOTE: If bleeding occurs while you are inserting the catheter into IVC, then you should try to insert the new catheter into a portal vein.
    15. Remove the needle of the catheter and control the position of the cannula to avoid getting out of IVC, then connect 24 G cannula with perfusion tube by using the connector.
      NOTE: Avoid the presence of air bubbles before starting the perfusion.
    16. Start the perfusion of the liver with warm HBSS (-) at a flow rate of 4 mL/min and cut off the splenic vein quickly to drain out the internal blood by using scissors.
      NOTE: If the cannulation is successful, the liver will begin to blanch due to the backflow distribution of the buffer from IVC to hepatic veins. The portal vein is formed by the union of splenic and superior mesenteric veins; therefore, it is quite safe to cut the splenic vein since it is big and distal from the liver.
    17. Continue perfusion with 35 mL of warm HBSS (-).
      NOTE: The liver will become pale in color if the perfusion is adequate, and the mouse still alive under anesthesia at this stage.
  3. Digestion and extraction
    1. Perfuse the mouse liver with warm 35 mL of HBSS (+) including Collagenase Type X at a flow rate of 4 mL/min.
    2. Every few minutes, clamp the splenic vein by using forceps for ~10 s to allow the entire volume of HBSS (+) to diffuse into all anatomical parts of the liver.
      NOTE: Liver begins to swell after clamping the splenic vein due to buffer congestion.
    3. Pour 5 mL of prepared warm HBSS (+) with Collagenase Type X into 100 mm Petri dish at the end of the perfusion process.
    4. Cut off the perfused liver from the rest of the body before stopping the pump to avoid the backflow of blood into the liver, remove the gallbladder by forceps, and then wipe the liver gently with a paper towel to remove the blood.
    5. Transfer the liver to the Petri dish which contains 5 mL of prepared warm HBSS (+) and remove the liver capsule gently using straight-tipped forceps.
      NOTE: The liver capsule is a thin layer of connective tissue that surrounding the liver.
    6. Disperse the parenchymal tissue carefully by using the forceps. Add 15 mL cold DMEM to the Petri dish.
      NOTE: The medium will turn cloudy due to parenchymal cells if the liver is well digested.
    7. Shake the torn liver gently to release the residual parenchymal cells into the medium and add another 15 mL cold DMEM to the Petri dish to get the rest of the cells.
    8. Transfer DMEM with the dissolved liver through 100 μm cell strainer into a 50 mL conical tube and keep in ice.
  4. Purification
    1. Centrifuge the cell suspension at 60 x g for 2 min at 4 °C, carefully remove the supernatant by vacuum aspiration and resuspend the pellet in 10 mL DMEM.
    2. Add resuspended pellet as a thin layer on the top of 40% density gradient buffer, and centrifuge at 800 x g for 10 min without brake at 4 °C.
    3. Carefully remove supernatant including the upper layer (DMEM) and the middle layer (dead or non-parenchymal cells), but not the lower layer (Pellet: primary parenchymal hepatocytes).
    4. Resuspend the primary cells with 2–3 mL William’s medium E, and transfer to the new 50 mL tube to avoid contamination of dead cells on the wall of the tube.
    5. Add 7-8 mL William’s medium E, mix gently, and centrifuge at 800 x g for 1 min at 4 °C.
    6. Carefully remove supernatant by vacuum aspiration and then resuspend pellet again with 10, 20 or 30 mL of William’s medium E (according to the size of the pellet).
    7. Count the cells by using an automated cell counter.
    8. Seed with William’s medium E, keep the plate in an incubator at 37 °C for 2-3 h, then replace the medium with10% FBS DMEM medium with Anti-Anti to remove the dead or unattached cells and for overnight incubation at 37 °C.
    9. Next day, primary mouse hepatocytes are ready for experimental use.

2. Chemo-selective Ligation Reaction Assay for the Detection of Nascent Protein Synthesis

  1. Metabolic labeling
    1. (Pre-treatment) Wash primary hepatocytes with 37 °C warm PBS twice and add the methionine-free DMEM medium for 30 min to deplete the cytoplasmic methionine reserves.
      NOTE: L-Azidohomoalanine, is an amino acid and analog to methionine, contains an azido moiety that can be incorporated into proteins during biosynthesis of cellular proteins so that depletion of cytoplasmic methionine will enhance the feeding and incorporation of L-Azidohomoalanine into newly synthesized proteins of cultured primary hepatocytes.
    2. (Treatment) Wash primary hepatocytes with 37 °C warm PBS twice and add the methionine-free DMEM medium with 25 μM AHA (L-Azidohomoalanine) for 4–6 h.
    3. Add any specific chemical (i.e. 10 μM rotenone) in the medium for 4–6 h in presence of AHA in order to investigate its effect on nascent protein expression levels.
    4. After incubation, remove the medium by vacuum aspiration and wash primary hepatocytes with cold PBS three times.
    5. Add 200 μL lysis buffer to the plate and incubate for 15-30 min on ice, then tilt the plate and take the whole cell lysate into 1.7 mL tube.
      NOTE: You should observe that cell lysate is very sticky during the cell harvesting.
    6. Sonicate the cell lysate on ice for 5 s three times by using a probe sonicator to solubilize the proteins and disperse the DNA.
    7. Vortex the cell lysate for 5 min, centrifuge the cell lysate at 21,130 x g for 10 min at 4 oC and then transfer the clear supernatant into a new tube. Measure the protein concentration twice.
      NOTE: At this stage, protein samples can be stored at -20 °C for two weeks if they are not immediately used.
  2. Labeling the Azide-modified nascent protein
    1. Prepare the components (A+B) by adding 60 μL of component (A) to component (B).
      NOTE: The solution turns to bright pink in color after adding component (A) to (B).
    2. Prepare the buffers (D) and (E) according to the protocol.
    3. Take 20–40 μg cell lysate and add 50 μL 2x reaction buffer (component A+B), then adjust the volume to 80 μL by adding DDW and vortex for 5 s.
    4. Add 5 μL CuSO4 (component C), and vortex for 5 s.
    5. Add 5 μL reaction buffer additive 1 (freshly prepared component D), then vortex for 5 s and wait for 3 min.
    6. Add 10 μL reconstituted reaction buffer additive 2 (component E), then vortex for 5 s and rotate samples for 20 min at 4 °C by using a multi-rotator machine.
      NOTE: The solution turns to bright orange in color after adding component (E). The samples should be protected from light during the rotation.
    7. Add 300 μL methanol and vortex for 5 s, add 75 μL chloroform and vortex for 5 s, then add 200 μL DDW and vortex for 5 s.
    8. Centrifuge the samples at 21,130 x g and 4 °C for 5 min, then discard the aqueous supernatant and keep the pellet.
    9. Add 250 μL methanol to the tube, vortex, and spin again for 5 min at 21,130 x g.
    10. Discard the supernatant, then cover the samples with the lint-free tissue and allow the pellets to air-dry for 15 min at room temperature.
  3. PAGE analysis and western blotting
    1. Solubilize the pellet in 20 μL of loading buffer without β-mercaptoethanol, vortex for 10 min, heat at 70 °C by using heat block for 10 min. Then load the samples into 10% SDS gel (1.5 mm) for Tetramethylrhodamine (TAMRA) detection.
    2. Detect AHA signal using a variable mode laser scanner for precise quantitation of nascent proteins.
    3. Wash the Gel with DDW for 15 min.
    4. Then, stain the 10% SDS gel with a fast and sensitive coomassie-dye reagent for 15–60 min to detect total proteins as a control.
    5. De-stain the Gel with DDW for 1–2 h and acquire the image by using a UV fluorescence imager.

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Results

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...

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Discussion

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...

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Disclosures

The authors indicate they have no conflicts of interest.

Acknowledgements

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.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
HEPES bufferFisher ScientificBP310-500
D-glucoseFisher ScientificD16-500
Ethylene glycol-bis(β-aminoethyl ether)-tetraacetic acidAmericanBioAB00505-00025
Antibiotic-Antimycotic (100x)Gibco15240-062
HBSS (10x) no calcium, magnesium, phenol redGibco14185-052
Calcium Chloride Dihydrate (CaCl2.2H2O)Fisher ScientificC79-500
Density gradient bufferGE Healthcare17-0891-02
DMEM (Dulbecco's Modified Eagle Medium) low glucose, pyruvateGibco11885-084
Fetal Bovine SerumHycloneSH30910.03
Phosphate Buffered Saline (PBS) (1x)Gibco1897141
Williams medium E, no glutamineGibco12551-032
L-alanyl-L-glutamine dipeptide supplementGibco35050-061
Collagenase Type XWako Pure Chemical Industries039-17864
Perfusion pumpCole-ParmerMasterflex L/SEquipment
IV administration setEXELINT29081Equipment
A water bathREVSCIRS-PB-200Equipment
Tube heaterFisher ScientificIsotempEquipment
EthanolDecon Lab, Inc0-39613
IsofluranePHOENIX10250
Autoclaved Cotton TipsFisherbrand23-400-124
100 mm Petri DishTPP93100
Connector (Male Luer Lock Ring)Cole-Parmer instrumentEW-4551807
24 G cathetersTERUMOSurflo 24Gx3/4'
100 μm Filter (CELL STRAINERS)VWR10199-658
15 mL conical-bottom centrifuge tubesVWR89039-666
50 mL conical-bottom centrifuge tubesVWR89039-658
Chemoselective ligation reaction PROTEIN ANALYSIS DETECTION KIT, TAMRA ALKYNEInvitrogenC33370
AHA (L-azidohomoalanine)InvitrogenC10102
DMEM (methionine free)Gibco21013024
L-Cystine DihydrochlorideSIGMAC2526
Laemmli sample bufferBioRad161-0737
Protease Inhibitor CocktailSIGMAP9599
SDS solution (20%)BioRad161-0418
Tris-HCL (1M)American BioanalyticalAB14044-01000
Phosphatase Inhibitor CocktailSIGMAP5726
Protein concentration measuring Kit (Bovin Serum Albumin-BSA)BioRad500-0207
6-well tissue culture plateTPP92006
Digital HeatblockVWR12621-092Equipment
Multi-RotatorGrant-bioPTR-60Equipment
Ultrasonic SonicatorCole-ParmerGE130PBEquipment
Standard Heavy-Duty Vortex MixerVWR97043-566Equipment
A variable mode laser scannerGE Healthcare Life ScienceFLA 9500Equipment
Coomassie-dye reagentThermo Scientific24594
Inverted microscopeOlympusCKX53Equipment
Western Blotting apparatusBioRad1658004Equipment
CentrifugeEppendorf5424REquipment
Automated cell counterBioRadTC20Equipment
FluorChem R systemproteinsimple-Equipment
p-Ampka (T172) antibodyCell signaling2535
Total-AMPK antibodyCell signaling5832
Albumin antibodyCell signaling4929
beta actin antibodySanta Cruzsc-130656
Fine scissors and forceps

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Hepatocyte IsolationCollagenase PerfusionDensity Gradient SeparationNon radioactive LabelingAHA DetectionWestern Blot AnalysisAlbumin ExpressionAMP activated Protein Kinase
Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method
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