קביעת חומצות שומן החמצון וLipogenesis בעכבר העיקרי hepatocytes
Summary
lipogenesis נובו דה וחמצון חומצת β-השומן מהווים מסלולי מפתח חילוף חומרים בhepatocyte, מסלולים המוטרדים בכמה הפרעות מטבוליות, כוללים מחלת כבד שומני. כאן אנו מדגימים בידוד של hepatocytes העיקרי עכבר ולתאר כימות של חמצון חומצת β-השומן וlipogenesis.
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
הזמן מהקרבה לזלוף צריך להיות פחות מ 3 דקות לזלוף אידיאלי ועיכול collagenase של הכבד. ברגע שזלוף עם זלוף בינוני הוא יזם, הכבד צריך לשנות באופן מיידי את מראה למאדום לחיוורים. לאחר כ 10 דקות של דגירה עם LDM, הכבד יופיע נפוח ורודה. במקרה שזלוף אינו מספיק, בכבד לא יכול להציג שינויים א…
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 |
Referências
- Clark, J. M., Brancati, F. L., Diehl, A. M. The prevalence and etiology of elevated aminotransferase levels in the United States. The American journal of gastroenterology. 98, 960-967 (2003).
- Lazo, M., et al. Prevalence of nonalcoholic Fatty liver disease in the United States: the third national health and nutrition examination survey, 1988-1994. American journal of epidemiology. 178, 38-45 (2013).
- Angulo, P. Nonalcoholic fatty liver disease. The New England journal of medicine. 346, 1221-1231 (2002).
- Adams, L. A., et al. The natural history of nonalcoholic fatty liver disease: a population-based cohort study. Gastroenterology. 129, 113-121 (2005).
- Feldstein, A. E., et al. Hepatocyte apoptosis and fas expression are prominent features of human nonalcoholic steatohepatitis. Gastroenterology. 125, 437-443 (2003).
- Day, C. P., James, O. F. Steatohepatitis: a tale of two ‘hits’. Gastroenterology. 114, 842-845 (1998).
- Donnelly, K. L., et al. Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease. The Journal of clinical investigation. 115, 1343-1351 (2005).
- Lambert, J. E., Ramos-Roman, M. A., Browning, J. D., Parks, E. J. Increased de novo lipogenesis is a distinct characteristic of individuals with nonalcoholic fatty liver disease. Gastroenterology. 146, 726-735 (2014).
- Parks, E. J., Hellerstein, M. K. Thematic review series: patient-oriented research. Recent advances in liver triacylglycerol and fatty acid metabolism using stable isotope labeling techniques. Journal of lipid research. 47, 1651-1660 (2006).
- Befroy, D. E., et al. Direct assessment of hepatic mitochondrial oxidative and anaplerotic fluxes in humans using dynamic 13C magnetic resonance spectroscopy. Nature medicine. 20, 98-102 (2014).
- Goncalves, L. A., Vigario, A. M., Penha-Goncalves, C. Improved isolation of murine hepatocytes for in vitro malaria liver stage studies. Malaria journal. 6, 169 (2007).
