|Home | About | Journals | Submit | Contact Us | Français|
Very long chain fatty acids confer functional diversity on cells by variations in their chain length and degree of unsaturation. Microsomal fatty acid elongation represents the major pathway for determining the chain length of saturated, monounsaturated, and polyunsaturated fatty acids in cellular lipids. The overall reaction for fatty acid elongation involves four enzymes and utilizes malonyl CoA, NADPH, and fatty acyl CoA as substrates. While the fundamental pathway and its requirements have been known for many years, recent advances have revealed a family of enzymes involved in the first step of the reaction, i.e., the condensation reaction. Seven fatty acid elongase subtypes (Elovl #1–7) have been identified in the mouse, rat, and human genomes. These enzymes determine the rate of overall fatty acid elongation. Moreover, these enzymes also display differential substrate specificity, tissue distribution, and regulation, making them important regulators of cellular lipid composition as well as specific cellular functions. Herein, methods are described to measure elongase activity, analyze elongation products, and alter cellular elongase expression.
Fatty acid elongation occurs in three cellular compartments: the cytosol, mitochondria, and endoplasmic reticulum (microsomes). In the cytosol, fatty acid elongation is part of de novo lipogenesis and involves acetyl-CoA carboxylase and fatty acid synthase. Fatty acid synthase utilizes acetyl CoA and malonyl CoA to elongate fatty acids by two carbons. The primary end product of de novo lipogenesis is palmitate (16:0) and to a lesser extent myristate (14:0) and stearate (18:0). Fatty acid elongation also occurs in mitochondria and utilizes an enoyl-CoA reductase plus acetyl CoA and fatty acyl CoA as substrates. This site appears to be a minor pathway for fatty acid elongation in eukaryotic cells (1). Microsomal fatty acid elongation is considered the predominant pathway for elongating fatty acids 12-carbons and longer (2–10). This pathway utilizes fatty acids derived from endogenous pathways, like de novo lipogenesis, as well as exogenous fatty acids derived from the diet.
The overall pathway for microsomal fatty acid elongation involves four enzymes and fatty acyl CoA, malonyl CoA, and NADPH as substrates (Fig. 1). In step 1, a 3-keto acyl-CoA synthase catalyzes the condensation of malonyl CoA with a fatty acyl-CoA precursor. In step 2, a 3-keto acyl-CoA reductase reduces the resulting 3-keto intermediate. Step 3 involves the dehydration of the 3-hydroxy species; this reaction is catalyzed by a 3-hydroxy acyl-CoA dehydratase. In step 4, reduction of the step 3 product is catalyzed by trans-2,3-enoyl-CoA reductase (2, 9). Cloning and genomic analysis have identified seven distinct 3-keto acyl-CoA synthases, i.e., fatty acid condensing enzymes, in mouse, rat, and human genomes (http://www.ensemble.org) (3, 4). These enzymes are called elongases and have been given the designation Elovl for elongation of very long fatty acids (Elovl 1–7). The elongase subtypes, their mouse accession # and alias are: Elovl-1: NM_019422, SSC1; Elovl-2: AF170908, SSC2; Elovl-3: BC016468, CIG30; Elovl-4: NM_148941, Elo4; Elovl-5: NM_134255, FAE1; Elovl-6: AY053453, LCE or FACE; Elovl-7: BAB31310, 9130013K24RIK. The two reductases, 3-keto acyl-CoA reductase and trans-2,3-enoyl-CoA reductase, have been cloned from human, mouse, and yeast (9, 11). The dehydratase, however, has not been cloned.
Fatty acyl-CoA substrate specificity and rate of fatty acid elongation is determined by the first step in the pathway, i.e., the activity of the condensing enzyme and not the reductases or dehydratase (1, 9). While Elovl-1, Elovl-3, and Elovl-6 elongate saturated and monounsaturated fatty acids (3, 4, 12). Elovl-2, Elovl-4, and Elovl-5 elongate polyunsaturated fatty acids. Elovl-5 also elongates some monounsaturated fatty acids, like palmitoleic acid (8). Elovl-5 specifically elongates γ-linolenoyl-CoA (18:3,n-6 CoA), while Elovl-2 specifically elongates 22-carbon PUFA (3, 4, 8). Substrates for Elovl-7 have not been described. The elongases (Elovl) are expressed differentially in mammalian tissues (3, 4). For example, five elongases are expressed in rat and mouse liver, including Elovl-1, -2, -3, -5, -6 (6, 7, 13). In contrast, the heart expresses Elovl-1, -5, and -6, but not Elovl-2 (14). These enzymes are also regulated by diet, hormones, during development, and in chronic disease (3, 7–9, 12). As such, changes in their activity impact cellular lipid composition.
Fatty acid elongases function with fatty acid desaturases to generate many of the long chain mono and polyunsaturated fatty acid assimilated into cellular lipids. Elovl-6 and Δ9-desaturase generate 18-carbon monounsaturated fatty acids like oleic acid (8, 12), while Elovl-2 and Elovl-5 along with Δ5 and Δ6 desaturases generate the end products of n-6 and n-3 synthesis, i.e., arachidonic and docosahexaenoic acid (3, 4, 8, 15).
This chapter describes several methods to study elongase expression and function in cells and tissues. First is a detailed description of methods to measure fatty acid elongase activity in rat and mouse liver microsomal preparations. Second is an approach to measure fatty acid elongation in cultured cells, like rat primary hepatocytes. Third is an approach to modify the expression of fatty acid elongases in cultured cells and mouse liver.
The fatty acid elongase reaction is carried out in 1.5–1.8-ml screw-capped microfuge tubes (12).
|100 mM KH2PO4, pH 6.5||50 mM||50 μl|
|1 mM rotenone (Sigma)||5 μM||0.5 μl|
|10 mM malonyl CoA (Sigma)||60 μM||0.6 μl|
|10 mCi/ml 14C-malonyl CoA (Perkin Elmer)||15 nCi||1.5 μl|
|10 mM Fatty acyl CoA (in 50 mM KH2PO4, pH 5.8) (Avanti Polar Lipids)||40 μM||0.5 μl|
|22 mM NADPH (Sigma) (made fresh each week in 50mM KH2PO4, pH 5.8)||1 mM||5 μl|
|400-μM bovine serum albumin (fatty acid and endotoxin free in HOH, Probumin from Celliance, Cat# 820473; Kankakee, Ill).||20 μM||5 μl|
|Microsomal protein||50 μg/0.1 ml||50 μg|
Adjust final volume to 100 μl with water. Mix and incubate 37°C. for 20 min.
Stop the reaction with the addition of 100 μl of 5 M KOH-10% methanol. Incubate for 1 h at 65°C. This step will hydrolyze (saponify) the ester bond between the fatty acid and coenzyme A. Stop the saponification step by adding 100 μl of 5 M HCl and 100 μl of ethanol to each reaction. Extract fatty acids by adding 750 μl of hexane–acetic acid mix (98% hexane–2% glacial acetic acid), vortex vigorously for ~1 min. Separate the aqueous (lower) and organic (upper) phases by centrifugation in a microfuge (5 min at maximum speed). Transfer upper phase (hexane phase) to a scintillation vial. Repeat hexane extraction and combine the hexane supernatants. Add 5 ml of scintillation cocktail (Ultima-Gold, Perkin Elmer) and count radioactivity.
Several important controls should be run for each study. First, NADPH is required for microsomal fatty acid elongation. In a control reaction, add all the reagents except NADPH. Radioactivity recovered in the hexane–acetic acid phase from this reaction represents background 14C radioactivity. Second, add 1.5 μl of 14C-malonyl CoA directly to the scintillation vial. Once background subtractions are made, this will represent 100% of 14C. Since the reaction contains 6 nmol of malonyl CoA, a simple % calculation of total radioactivity appearing in the hexane–acetic acid phase will indicate the nmoles of malonyl CoA transferred to a fatty acid.
Express the results as nmoles/mg microsomal protein. When using 18:3(n-6) CoA as substrate and rat liver microsomes, expect ~3–5 nmol of substrate (malonyl CoA) converted to product (fatty acid)/mg protein/20 min reaction.
With few exceptions, the product of the fatty acid elongase reaction is the result of the action of two or more fatty acid elongases. For example, 16:0-CoA is a substrate for Elovl1, Elovl-3, and Elovl-6, while 20:4(n6)-CoA is a substrate for Elovl-2 and Elovl-5. It is possible to measure Elovl-2 and Elovl-5 specifically using 22:5(n3)-CoA and 18:3(n6)-CoA as substrates, respectively. As such, an important step in elongase analysis is to establish the elongase subtype expression in cells under study. This can be accomplished using quantitative reverse transcriptase-polymerase chain reaction. Primers for several mouse, rat, and human elongases have been reported elsewhere (8).
Because the commercial availability of fatty acyl CoAs is limited, nonesterified fatty acids can be substituted in the reaction. For details of this modification of the elongase assay, see Note 1.
Addition of nonesterified fatty acids to cells leads to their rapid uptake, metabolism, and assimilation into complex lipids. Many fatty acids undergo elongation and desaturation. The products of these reactions can be monitored using a combination of 14C-labeled fatty acids, cell culture, total lipid extraction, and analysis of saponified fatty acids by reverse phase-high performance liquid chromatography (RP-HPLC) coupled to a flow-through β-scintillation counter (8, 15, 16).
Rat primary hepatocytes are treated with 14C-fatty acids to assess fatty acid elongation. Primary hepatocytes are prepared from Teklad chow-fed (ad lib) male Spraque–Dawley rats and cultured on 6-well BioCoat (type 1 collagen) plates (Beckon Dickinson, Belford, MA.) (16). Cells are incubated in Williams E medium (Invitrogen) containing 25 mM glucose, 1 microM insulin (Invitrogen, Carlsbad, CA); 25 μM bovine serum albumin (endotoxin and fatty acid free albumin from Celliance, Kankakee, Ill), and 100 μM 14C-fatty acid [1.6 Ci/mol] (16). See Note 2 regarding fatty acid treatment of other cell types.
Total lipid extraction and analysis of fatty acids is carried out after treating cells with 14C-fatty acids. At harvest, remove the media from cells and wash cells once with cold phosphate-buffered saline (PBS) containing 200 μM BSA followed by a wash with PBS alone. After removal of the PBS, resuspend cells in 0.5 ml of 40% methanol + 0.1N HCl; use a 25-mm cell scraper (Sardstedt) to remove cells from the plastic culture dish. Transfer the resuspended cells to 15-ml glass centrifuge (Kimble HS No. 45500.15) tube (see Note 3). If a protein assay is required, then 2 μl of the cell suspension can be taken for a protein assay (Bio-Rad, Hercules, CA). Add 2 ml of chloroform:methanol (2:1) plus 1 mM butylated hydroxytoluene (BHT) (Sigma-Aldrich) to the cell suspension and vortex carefully, avoid loss of sample. After thorough mixing, the sample is centrifuged in a JA20.1 rotor at 5,000 rpm (3220 × g) using a Beckman Avanti refrigerated (4°C) centrifuge. The organic (lower) phase is recovered and transferred to a 12-ml screw-cap tube (Fisher Pyrex #9825). The aqueous phase and protein interface is re-extracted with 2-ml chloroform as before. After centrifugation, the organic phase is transferred to the same 12-ml screw-capped tube. The organic solvents are removed by a constant stream of nitrogen using a nitrogen evaporator (Organomation Associates, Inc). All the moisture is removed using a microcentrifuge evaporator (CentriVap + cold and organic solvent traps, Labconco) overnight.
The next day, dissolve lipids in 500 μl of chloroform containing 1 mM BHT. Lipids are transferred to storage vials (12 × 35 mm, Fisher cat # 03338AA), labeled, and stored at −80°C. At this point, total lipid extracts can be fractionated by thin-layer chromatography to assess the distribution of 14C in complex lipids or saponified for analysis of fatty acid by RP-HPLC (15).
An alternative method for analysis of elongation products involves treating cells with saponification buffer (0.4N NaOH in 80% methanol). In this method, cells grown in 6-well plates are resuspended in 500 μl of saponification buffer per well of a 6-well plate. Transfer the resuspended cells to a 12-ml pyrex tube, capped, and heated as described above (Subheading 3.2.3, step 2). Follow steps 2–7 to prepare fatty acids for RP-HPLC.
The equipment used for RP-HPLC includes a Waters 600 controller (Milford, MA), Waters UV/Vis absorbance monitor [model 2487], Waters evaporative light scatter detector [model 2420], and a β-RAM flow through β-scintillation counter (IN/US Systems, Inc., Tampa, Fl). The reverse phase column is a YMC J’Sphere (ODS-H80, 4 μm, 8 nm; 250 × 4.6 mm I.D.) (Waters Corporation). Eluate from the column passes through the UV detector, exits and is split 1:1 for flow into the evaporative light scatter detector and the flow through β-scintillation counter. The scintillation cocktail for the IN/US unit is Inflow 2:1 (IN/US Systems, Inc.). The ratio of eluant to scintillation cocktail is 2:1. The software for data collection and analysis is Winflo (IN/US Systems, Inc.).
Samples (20 microl) are injected and fatty acids are eluted from the column using a mobile phase consisting of a linear gradient starting at 77.5% acetonitrile + 22.4% water + 0.1% acetic acid and ending at 99.9% acetonitrile + 0.1% acetic acid over 90 min with a flow rate of 1.0 ml/min (see Note 4). Buffers are sparged with helium prior to and during HPLC runs. Fatty acids are detected using UV absorbance and evaporative light scatter (see Note 5). Waters 2487 dual absorbance monitor parameters are: 192 nm and a gain of 0.5. The parameters for the Waters 2420 light scatter detector are: gain = 100; PSI = 5 psi, ultrapure nitrogen; nebulizer tube temperature = 60% or 36°C; drift tube temperature = 45°C. While UV absorbance detects only unsaturated fatty acids, evaporative light scatter detects saturated and unsaturated fatty acids. The inline β-scintillation counter detects 14C-labeled fatty acids (8, 16). In some cases, nonradioactive fatty acids can be used to treat cells (see Note 6). Additional verification of fatty acid elongation products was established by gas chromatography–mass spectrometry at the mass spectrometry facility at Michigan State University (http://genomics.msu.edu/mass_spec/index.html). The equipment used in these studies was sufficient to establish the chain length and the number of double bonds (16). In addition, the chromatograms of 14C-label fatty acids were compared to the chromatograms of unlabeled fatty acid standards. Fatty acid standards used in our studies include: 14:0; 16:0; 18:0; 16:1,n-7; 18:1,n-9; 19:1,n- (recovery standard); 18:2,n-6; 18:3,n-3, 18:3,n-6; 20:3,n-6, 20:3,n-9, 20:4,n-6, 20:5,n-3; 22:5,n-3; 22:6,n-3. Each fatty acid is at 100 μM in methanol. All fatty acids are obtained from Nu-Chek Prep (Elysian, MN).
Mouse models of ablated expression of Elovl-3 and Elovl-6 genes have been reported (6, 17). The phenotype of these animals indicates that Elovl-3 and Elovl-6 play important roles in skin lipid composition and whole body insulin action, respectively. Elovl-2 has been overexpressed in mouse adipocyte cell lines and appears to influence triglyceride metabolism (18). Elovl-5 has been over-expressed in mouse liver (15); elevated Elovl-5 expression affects multiple pathways involved in hepatic lipid and carbohydrate composition (15). Such studies indicated that fatty acid elongases have effects on cellular functions that go beyond effects on cellular lipid composition, per se.
A key requirement for assessing elongase functions in cells and in vivo is to alter the expression of the enzyme in nearly all cells in culture or in a tissue. Our approach to alter elongase expression in cells and liver has used recombinant adenoviruses expressing specific elongases (condensing enzyme). This method allows for robust expression of the elongase in nearly every cell in culture and uniform expression in hepatic parenchyma cells in liver (15). Under these conditions, it is possible to assess the impact of these enzymes on cellular lipid composition and function. Methods to prepare recombinant adenoviruses expressing elongases and their use in cells or in vivo have been described elsewhere (8, 15). Changes in cell/tissue lipid composition are assessed by the methods described above for monitoring elongase activity and fatty acid composition.
Replication defective recombinant adenoviruses are a biohazard. As such, the use of these reagents require a biosafety level-2 (BSL2) for containment of both cell culture and animal infection studies. Adenoviruses generated in our laboratory are derivatives of the Ad-Easy recombinant adenovirus (Stratagene). Standard cloning protocols are followed to generate these viruses. Typically, coding regions of specific enzymes are generated by reverse transcriptase-polymerase chain reaction and inserted into the pShuttle-CMV vector (Stratagene). Cloning of cDNA for Elovl-2, Elovl-5, and Elovl-6 was described previously (7). The coding region for each transcript was ligated into Ad-Easy XL adenoviral vector system (Stratagene), recombined in BJ5183 cells, and propagated in XL10 Gold ultracompetent cells. Ad-DNA was packaged into adenoviral particles in Ad-293 cells. The resultant adenovirus was amplified in Ad-293 cells. Viral lysates are cesium purified twice and titered using the Adeno-X Rapid Titer Kit (Clontech).
Rat primary hepatocytes are infected with recombinant adenovirus using the following protocol. Rat primary hepatocytes are prepared as described (Subheading 3.2.1). Confluent primary hepatocytes are infected with virus at 5–10 plaque-forming units[PFU]/cell). When an adenovirus expressing green fluorescent protein (GFP) is used as a control for infection, greater than 80% of primary hepatocytes express functional protein at the 5–10 PFU/cell level. Under these infection conditions, fatty acid elongase activity increases twofold to sixfold (8, 15). Typically, cells are infected overnight; the next day cells are ready to assess the effects of altered elongase expression on fatty acid elongase activity (Subheading 3.1) or fatty acid elongation in cells (Subheading 3.2).
C57BL/6 mice are infected with recombinant adenovirus using the following protocol. Male C57BL/6 (Charles River Laboratories or Jackson Labs) mice (20–22 g; ~2 months of age) are used for studies with recombinant adenovirus in vivo. Mice are maintained on a Harlan–Teklad (#8640) diet ad lib throughout all studies. Mice are injected with recombinant adenovirus by a retro-orbital route at a dose of 2 × 1010 viral particles (cesium-purified)/mouse while under isoflurane anesthesia. A BD ultrafine II insulin syringe (short needle) is used for the injection. Mice injected with phosphate-buffered saline + 1.0% glucose served as a “Saline” control. Control virus infection uses a recombinant adenovirus expressing firefly luciferase (Luc) (15). In our experience, the Ad-Luc virus serves as an excellent control for infection. In contrast, Ad-GFP has been found to promote hepatotoxicity as reflected by elevated levels of plasma alanine aminotransferase activity. Body weight, food, and water intake is monitored daily. Typically animals displayed no adverse effects from the saline or adenovirus injection and gained weight equally for the duration of the study (5 days). Mice are euthanized on day 5 after infection for the collection of tissues and blood.
The advantage of in vivo studies is that it allows for a thorough examination of effects of altered hepatic elongase expression on liver and plasma lipid composition as well as multiple hepatic functions, like gene expression, cell signaling and glycogen, triglyceride, and cholesterol content. Such studies have provided novel insight into the impact altered elongase expression has on multiple pathways involved in lipid and carbohydrate metabolism (15). A limitation of this in vivo approach, however, is that recombinant adenoviruses infect hepatic cells, including Kupffer and parenchymal cells. Following infection of mice with recombinant adenovirus, we have found ample expression of the elongase in liver, but no change in elongase expression in nonhepatic tissues, like heart, kidney, and spleen (15). A second limitation is that expression of the transgene from the infecting adenovirus persists for only a week or so. Other methods for longer expression are available using other viral vectors, e.g., adeno-associated virus or helper-dependent adenovirus.
An example of the elongation of a fatty acid in rat primary hepatocytes is illustrated in Fig. 2. In this study, primary hepatocytes were treated with 14C-20:4,n-6 as described in Subheading 3.2. After treating cells with the fatty acid, cells were harvested and lipids extracted. Saponified lipids were fractionated by RP-HPLC. The chromatogram in Fig. 2 illustrates the separation of 14C-fatty acids. Arachidonic acid (14C-20:4,n-6) was added to the cells and both 14C-20:4,n-6 and its elongation product, adrenic acid (14C-22;4,n-6) are recovered following lipid extraction and saponification. In this study, ~30% of the total 14C-fatty acid recovered from the cells was adrenic acid (22:4,n-6) indicating that the substrate, (20:4,n-6) was elongated in rat primary hepatocytes.
In an in vivo study, Elovl-5 was overexpressed in C57BL/6 mouse liver by using the recombinant adenovirus approach described in Subheading 3.3.5. Hepatic Elovl-5 enzyme activity was elevated threefold and a product of Elovl-5 action, i.e., di-homo-g-linolenic acid (20:3,n-6), was elevated greater than two-fold in both liver and plasma (15). In addition, the expression of several genes targeted by the fatty acid regulated transcription factor, PPARα, was suppressed by elevated Elovl-5 expression. The outcome of these studies indicates that changes in hepatic Elovl-5 activity affects both hepatic and plasma lipids as well as hepatic gene expression (15).
|Components||Final concentration||Amount/volume added|
|50 mM CoASH||100 μM||0.5 μl|
|42 mM ATP||1 mM||2.4 μl|
|1 M MgCl2||1 mM||0.1 μl|
|100 mM fatty acid (in Methanol) (Nu-Chek-Prep, Elysian, MN)||20 μM||0.25 μl|
|100 mM NaOH||20 μM||0.25 μl|
This research was supported by the National Institutes of Health, DK43220. I would like to thank Dr. Moises Torres-Gonzalez and Sasmita Tripathy for critical reading of this manuscript.