マウス初代肝細胞における脂肪酸酸化および脂質生成の決意
Summary
デノボ脂質生成およびβ脂肪酸酸化は、肝細胞に脂肪肝疾患を含むいくつかの代謝障害に摂動された経路を主要な代謝経路を構成しています。ここでは、マウス初代肝細胞の単離を実証し、β – 脂肪酸の酸化および脂質生成の定量化について説明します。
Abstract
Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders.
In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via β-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying β-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling.
Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of β-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.
Introduction
Non-alcoholic fatty liver disease is one of the leading causes of liver disease in Westernized cultures1,2. Lipid accumulation within the liver is associated with cell death, fibrosis, and liver failure via yet unknown mechanisms3-6. In fatty liver disease, hepatocyte-mediated β-fatty acid oxidation and de novo lipogenesis are important determinants of net lipid accumulation7,8. This article will, therefore, focus on hepatocyte isolation, followed by quantification of β-fatty acid oxidation and de novo lipogenesis.
Numerous methodologies have been developed to interrogate hepatocyte lipid metabolism. Though it is possible to measure metabolism of fat in vivo using stable isotopes9,10, these methods are costly, and require large numbers of animals. Additionally, the ability to investigate the effect of exogenous chemicals is limited due to the nature of in vivo experimentation. In contrast, the isolation of primary hepatocytes from mouse liver provides an affordable avenue to pursue11. Furthermore, studying hepatocytes in culture allows investigators to study the effects of varying chemicals on lipid processing while circumventing the difficulties of in vivo experimentation. Finally, isolated hepatocytes avoid any confounding from varying genetics since they are derived from the liver of a single animal.
Here we isolate and culture of hepatocytes, and we measure β-fatty acid oxidation and de novo lipogenesis, using radiolabeled palmitate. The protocol detailed below is straight forward, effective, and reproducible.
Protocol
Representative Results
Discussion
犠牲から灌流までの時間は、肝臓の理想的な灌流およびコラゲナーゼ消化のために3分未満でなければなりません。灌流培地で灌流が開始されると、肝臓はすぐに淡赤からに外観を変更する必要があります。 LDMとのインキュベーションの約10分後、肝臓が腫れてピンク色に表示されます。潅流が不十分である場合には、肝臓はこれらの変化を示さないことがあり、これは典型的にはより低い肝…
Declarações
The authors have nothing to disclose.
Acknowledgements
We would like to acknowledge Susan Gray and Umadevi Chalasani for their help with technical aspects of the hepatocyte isolation protocol. This work was supported by NIDDK grant 5R01DK089185 (to M.P. Cooper) and the DERC Pilot and Feasibility Program at UMMS (to M.P. Cooper).
Materials
| Liver Perfusion Medium | Life Technologies | 17701038 | |
| Liver Digest Medium | Life Technologies | 17703034 | Aliquot and store at -20 °C |
| PBS | Corning | 21-040-CV | |
| 10X DPBS | Corning | 46-013-CM | |
| DMEM | Corning | 10-017-CV | |
| FBS | Life Technologies | 26140079 | |
| Collagen | Life Technologies | A1048301 | |
| Colloidal silica coated with polyvinylpyrrolidone | GE Life Sciences | 17-0891-01 | |
| Sodium Pyruvate | Cellgro | 25-000-CI | |
| Penicillin / Streptomycin | Cellgro | 30-001-CI | |
| Insulin | Sigma | I0516-5ML | |
| Dexamethasone | Sigma | D2915-100MG | |
| Albumin (BSA), Fraction V | MP Biomedicals | 103703 | |
| 24-Well Culture Dish | Corning Falcon | 353047 | |
| Tygon S3 Tubing | Cole Parmer | 06460-34 | |
| Male Leur Lock to 200 Barb Connectors | Cole Parmer | 45518-00 | |
| 24G x 3/4" Catheter | SurFlo | SROX2419CA | |
| Perma-Hand Silk Suture | Ethicon | 683G | |
| Cell Strainer | Corning Falcon | 08-771-2 | |
| IsoTemp 3013HD Recirculating Water Bath | Fisher | 13-874-3 | |
| MasterFlex C/L Peristaltic Pump | MasterFlex | HV-77122-24 | |
| Microclamp | Roboz | RS-7438 | Pre-sterilize in autoclave |
| 5” Straight, Blunt-Blunt Operating Scissors | Roboz | RS-6810 | Pre-sterilize in autoclave |
| 24mm Blade Straight, Sharp-point Microdissecting Scissors | Roboz | RS-5912 | Pre-sterilize in autoclave |
| 4” 0.8mm Tip Microdissecting Forceps | Roboz | RS-5130 | Pre-sterilize in autoclave |
| 4” 0.8mm Tip Full Curve Microdissecting Forceps | Roboz | RS-5137 | Pre-sterilize in autoclave |
| 60 mL Syringe | Becton Dickinson | 309653 | |
| 50 mL conical tubes | Corning Falcon | 352070 | |
| BCA Protein Assay | Thermo Scientific | 23225 | |
| Biosafety Cabinet | |||
| CO2 Incubator | |||
| Serological pipets | |||
| 1000, 200, 20 μL pipet and tips |
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