Saturated Fatty Acids Induce Ceramide-associated Macrophage Cell Death

Summary

We illustrate a straight-forward method to derive murine primary macrophages from bone marrow cells and a simple method to prepare BSA-fatty acid conjugates. Then we demonstrate that saturated fatty acids can induce macrophage cell death, and such cell death is positively associated with cellular accumulation of ceramide levels.

Abstract

Macrophages highly express epidermal fatty acid-binding protein and adipose fatty acid-binding protein. They actively uptake saturated and unsaturated fatty acids, which might play a critical role in regulating their immune functions. Numerous studies have shown that various fatty acids, saturated or unsaturated, may possess different impacts on cell growth and function. However, the approaches used for fatty acid preparation vary, which may lead to non-physiological results. Serum albumin, a natural carrier for fatty acids in mammalian peripheral blood, is recommended for forming a conjugate complex with the sodium salt of fatty acids to study fatty acid function in mammalian cells, thus minimizing the toxicity of fatty acid soap. Thus, a simple, relatively quick heating and sonicating method is developed and presented here for BSA-fatty acid conjugate formation. We describe a protocol using saturated fatty acids, especially stearic acids to induce severe cell death in mouse bone-marrow derived macrophages. We further demonstrate that saturated fatty acid-induced cell death is positively associated with accumulated cellular ceramide levels. This method can be extended for studies of the impact of fatty acid on other mammalian cells.

Introduction

Fatty acids play a critical role in energy metabolism and in the synthesis of membrane phospholipids in different kinds of cells. Fatty acids have a low aqueous solubility. Appropriate preparation of fatty acid is of critical importance for studying the biological functions of fatty acids in mammalian cells. When fatty acids are prepared with ethanol, many fatty acids may show their toxic soap (detergent) effect on the cell membrane, even at relatively low concentrations¹. As a natural, major transporter for free fatty acids in the serum, serum albumin is considered a good carrier for fatty acid delivery in vitro for fatty acid functio....

Protocol

The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of University of Louisville.

1. Mouse Bone Marrow Derived Macrophages (BMDMs)

1. Euthanize a 6- to 8-week-old, healthy wild type mouse using CO₂. Pin it down to a foam board and spray it with 70% ethanol until it is soaked. Remove the tibia/femur bones with forceps and scissors. Put them in a petri dish with 5 mL 1x phosphate-buffered saline (PBS). Perform all procedures under sterile conditions.
2. Cut open both ends of the tibia/femur bone with a scissor in a sterile tissue culture hood. Flush the bone with a 25 G needle with PB....

Results

Obesity increases free fatty acid concentrations in serum. As professional phagocytes, macrophages actively take up fatty acids to maintain host homeostasis. During these processes, overloaded lipids may induce macrophage cell death. To this end, we cultured BMDMs in vitro with obese levels of dietary fatty acids and measured macrophage cell death using flow cytometric staining. Compared to the BSA control, saturated fatty acids, in particular stearic acids, induced significant c.......

Discussion

The proper preparation of fatty acid solution is of critical importance to study the biological function of fatty acids. The neutralization of fatty acid increases its solubility in aqueous solution. However, sodium salts of fatty acids, especially saturated fatty acids, are still of low solubility in water or PBS as we observed. One method is to use 95 - 100% ethanol to help dissolve fatty acid¹. Using this method, higher toxicity to cells may be observed even at lower concentrations for less tox.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CPX Ultrasonic Bath	Bransonic	Model 2800
Sodium palmitate (PA)	Nu-Chek Prep, Inc.	S-1109	M.W. 278
Sodium stearate (SA)	Nu-Chek Prep, Inc.	S-1111	M.W. 306
Bovine serum albumin (BSA), fatty acid-free	Fisher Scientific	9048-46-8
Mouse macrophage colony stimulating factor (mM-CSF)	Cell Signaling Technology,  Inc.	5228
RPMI 1640	VWR International	71002-878
Annexin V, Alexa Fluor 488 conjugate	Fisher Scientific	A13201
7-AAD	BD Biosciences	559925
Monoclonal anti-ceramide antibody (mouse IgM)	Sigma	C8104-50TST	Clone: MID 15B4
Goat Anti-Mouse IgM Antibody, µ chain, FITC conjugate	Sigma	AP128F
Fixation buffer	Biolegend	420801
Permeabilization buffer	Ebioscience	4307693
Red Blood Cell Lysis Buffer	Sigma	11814389001
Annexin V Binding Buffer	BD Biosciences	556454
L929 cells	ATCC	CCL-1
Corning Cell Lifter	Fisher Scientific	07-200-364
Note: M.W. is for molecular weight.