在小鼠原代肝细胞测定脂肪酸氧化和脂肪生成的
Summary
从头脂肪合成和β-脂肪酸氧化构成肝关键代谢途径，被扰动在几种代谢疾病，包括脂肪肝疾病途径。在这里，我们证明了小鼠原代肝细胞的分离和描述了β-脂肪酸氧化和脂肪生成量化。
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
从牺牲灌注时间应小于3分钟的理想灌注和肝脏的胶原酶消化。一旦灌注灌注中启动，肝应立即改变外观，从红色到浅。经过与LDM大约10分钟的潜伏期，会使肝脏出现浮肿和粉红色。倘灌注不足，肝脏可能不表现出这些变化，而这通常会导致较低的肝细胞的产率。
以下的洗涤步骤后，分离的肝细胞可以在电镀之前被存储数小时悬浮液在冰上。一旦镀金，培养肝细胞需要几个小?…
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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