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Department of Animal Science, University of California, Davis 95616
Corresponding author: A. L. Van Eenennaam; e-mail: alvaneenennaam{at}ucdavis.edu.
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ABSTRACT |
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-linolenic (ALA, 18:3n-3) essential fatty acids. These essential PUFA are generated by the action of desaturases that successively direct the conversion of monounsaturated fatty acids (MUFA) to PUFA. The cDNA coding sequences of the C. elegans 
12 and n-3 fatty acid desaturases were each placed under the control of separate constitutive eukaryotic promoters and simultaneously introduced into HC11 mouse mammary epithelial cells by adenoviral transduction. Phospholipids from transduced cells showed a significant decrease in the ratios of both MUFA:PUFA and n-6:n-3 fatty acids relative to control cultures. The fatty acid profile of transduced cellular phospholipids revealed significant decreases in MUFA and arachidonic acid (20:4n-6), and increases in LA, ALA, and eicosapentaenoic acid (20:5n-3). The fatty acid composition of triacylglycerols derived from transduced cells was similarly, but less dramatically, affected. These results demonstrate the functionality of C. elegans fatty acid desaturase enzymes in mammalian cells. Expression of these desaturases in livestock might act to counterbalance the saturating effect that rumen microbial biohydrogenation has on the fatty acid profile of ruminant products, and allow for the development of novel, land-based dietary sources of n-3 PUFA.
Key Words: polyunsaturated fatty acid • n-3 • fatty acid desaturase • transgenic
Abbreviation key: ALA = -linolenic acid, CoA = coenzyme A, FAT-1 = n-3 fatty acid desaturase, FAT-2 =12 fatty acid desaturase, LA = linoleic acid, MUFA = monounsaturated fatty acids, PUFA = polyunsaturated fatty acids, SFA = saturated fatty acids.
Long-chain polyunsaturated fatty acids (PUFA) play a particularly important role in fetal development and the maintenance of overall human health, either as a component of membrane phospholipids, or as precursors to various eicosanoids (Jump, 2002). The most abundant PUFA are often described as n-3 (omega-3) or n-6 (omega-6), which refers to the number of the first carbon with a double bond relative to the carbon at the methyl end of the molecule. Because n-3 and n-6 PUFA are not interconvertible in mammals, elevating tissue concentrations of n-3 PUFA relies on chronic dietary intake of fats rich in n-3 PUFA. It has been observed that the ratio of n-6:n-3 PUFA present in the diet of industrial societies has increased as a result of the greater consumption of vegetable oils rich in n-6 fatty acids and a reduced consumption of fish and plant sources of n-3 fatty acids. This imbalance has been linked to an increased risk of coronary heart disease and other human ailments (Connor, 2000; Simopoulos, 2004). According to current dietary customs, the consumption of fish is the most practical source of long chain n-3 PUFA and concern exists as to whether the current fish-based supply is adequate to meet future needs (Pauly et al., 2002). To supply the requirements of a growing population, long-chain n-3 PUFA sources that are renewable and sustainable need to be developed.
Animal products represent a large proportion of the Western diet and are an important source of protein. Cattle contribute 36.1% of all protein consumed, whereas fish supply only 5.5% of total protein (Smit et al., 1999). Beef and dairy products contain low levels of PUFA because of the extensive fatty acid biohydrogenation carried out by the rumen microbial population. Increasing the proportion of PUFA in ruminant products could have significant human health benefits (Visioli et al., 2000). One approach to achieve this goal has been to treat dietary feedstuffs with heat or chemicals to protect the PUFA from rumen biohydrogenation (Noakes et al., 1996). This approach has met with only limited success and remains dependent upon the provision of exogenous PUFA in the feedstuff. An alternative approach to develop novel land-based sources of PUFA would be to genetically engineer animals to produce their own desaturase enzymes to allow for de novo fatty acid desaturation.
Vertebrates possess the stearoyl coenzyme A (CoA)-desaturase enzyme required to synthesize monounsaturated fatty acids (MUFA) from saturated fatty acids (SFA). However, they lack the fatty acid desaturase enzymes required for the synthesis of linoleic acid (LA, 18:2n-6) and -linolenic acid (ALA, 18:3n-3), and are therefore dependent on dietary sources of these essential PUFA. The nematode Caenorhabditis elegans is able to synthesize both LA and ALA by virtue of an endogenous n-3 fatty acid desaturase (FAT-1) that recognizes a range of 18- and 20-carbon n-6 substrates (Spychalla et al., 1997) and a 12 fatty acid desaturase (FAT-2) that converts 16- and 18-carbon MUFA to n-6 fatty acids (Peyou-Ndi et al., 2000). We hypothesized that simultaneous expression of FAT-1 and FAT-2 in mammalian cells would enable the conversion of MUFA to n-3 PUFA and thus, the endogenous production of both LA and ALA essential fatty acids.
To test this hypothesis, FAT-1 and FAT-2 were constitutively expressed in HC11 mouse mammary epithelial cells. HC11 is a line of cells originally derived from COMMA-1D, an immortal cell line established from the mammary tissue of midpregnant BALB/c mice. This cell line can be grown using simplified cell culture conditions because it has no requirements for the addition of an exogenous extracellular matrix or co-cultivation with other cell types (Ball et al., 1988). Cells were maintained in growth medium consisting of RPMI+L-Glutamine (Gibco, Carlsbad, CA), supplemented with 8% heat-inactivated fetal calf serum (Gibco), 10 ng/mL epidermal growth factor (Sigma Chemical Co., St. Louis, MO), 5 µg/mL bovine insulin (Sigma Chemical Co.), and 50 µg/mL gentamicin (Gibco) in a humidified incubator at 37°C + 5% CO2 (Marte et al., 1994). Cells were subcultured at a ratio of 1:5 every 3 d. HC11 cells were plated at 2 x 106 cells per 60-mm Petri dish on the day before infection with the prepared adenovirus.
To construct the adenoviral expression vector, the C. elegans fat-1 (GenBank accession number L41807) and fat-2 (GenBank accession number NM_070159) cDNA sequences were placed under the control of the CMV and EF-1 alpha constitutive promoters in pBudCE4.1 (Invitrogen, Carlsbad, CA) respectively (Figure 1
), and subcloned into the Gateway pENTR 2B Vector (Invitrogen). A recombination reaction between the attL and attR attachment sites was performed to insert the desaturase genes into the pAd/CMV/V5/DEST Gateway Vector (Invitrogen) containing the human adenoviral gene, resulting in the pAd/fat-1/fat-2 construct.
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To produce the adenovirus, both pAd/fat-1/fat-2 and a control plasmid (pAd/CMV/V5-GW/lacZ, Invitrogen) were digested with PacI to expose left and right viral inverted terminal repeats. Fragments were purified with a QIAprep Spin Miniprep Kit (Qiagen, Valencia, CA). The 293A cells were plated in 6-well plates at 5 x 105 cells/well. The following day, at approximately 90% confluency, the cultures were transfected with pAd/fat-1/fat-2 or pAd/CMV/V5-GW/lacZ using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol with 3 µL of Lipofectamine 2000 and 1 µg of DNA per well. At 48 h post-transduction, the cells were transferred to a T-75 flask with complete growth media. Media were replaced every 3 d until cytopathic effects were observed, which was at approximately 12 d post-transduction. Approximately 18 d post-transduction, cytopathic effects had spread to 80% of the culture and plaques were visible. The cells and culture media were removed from the flasks and subjected to 3 rounds of freeze/thaw at –80°C (30 min) and 37°C (15 min). The cell lysate was centrifuged, and the supernatant containing the Ad/fat-1/fat-1 or Ad/lacZ viral lysate was aliquoted, stored at –80°C, and titered before use.
HC11 cells were infected with either the Ad/fat-1/fat-2 or the Ad/lacZ virus at a multiplicity of infection of 10. One culture transduced with Ad/lacZ virus was stained 3 d post-transduction using a ß-galactosidase staining kit (Mirus, Madison, WI) to determine the efficiency of transduction (Figure 2). The adenoviral system resulted in a very high efficiency (~95%) of transduction and was found to be much more effective as a means of gene delivery to HC11 cells than lipofection (data not shown).
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Statistical analysis of cellular fatty acid profiles from 6 experimental replicates per virus treatment (Ad/fat-1/fat-2 or the Ad/lacZ virus) was performed using a 1-way ANOVA. To examine the effect of the desaturase transgenes on the ratios of MUFA (16:1 + 18:1):PUFA (18:2 + 18:3 + 20:4 + 20:5) and n-6 (18:2 + 20:4):n-3 (18:3 + 20:5) fatty acids, the variance and covariance of the ratios were used to calculate the standard error of the difference between ratios (Cochran, 1977). A one-tail test of the hypothesis of the difference between the 2 ratios was performed using the calculated standard error: R –R' ±1.65 x SE(R –R') where R and R'represent the ratios being compared, SE(R –R') the standard error of the difference between the 2 ratios, and 1.65 = Z0.05.
Phospholipids from mammalian cells expressing FAT-1 and FAT-2 showed a significant (P <0.05) decrease in the ratios of both MUFA:PUFA (1.08 vs.1.81) and n-6:n-3 fatty acids (0.81 vs. 6.81) relative to the ratios found in control cultures expressing lacZ, respectively. The fatty acid profile of cells expressing FAT-1 and FAT-2 revealed an increase in LA, reflecting the known 12-desaturase activity of FAT-2; and both a decrease in arachidonic acid (20:4n-6) and increases in ALA and eicosapentaenoic acid (20:5n-3), reflecting the known n-3 desaturase activities of FAT-1 (Figure 3a). Although LA is a substrate for FAT-1, the decrease in LA due to desaturation by FAT-1 was likely overshadowed by the influx of LA from the desaturation of oleic acid by FAT-2. The fatty acid composition of triacylglycerols derived from transfected cells was similarly, but less dramatically, affected (Figure 3b).
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Unlike mammalian desaturases that act on fatty acyl-CoA triacylglycerol precursors (Pereira et al., 2003), FAT-1 and FAT-2 are hypothesized to be acyl-lipid desaturases. This may explain why the desaturases appeared to have a more immediate effect on the composition of phospholipid-bound acyl groups. This speculation is supported by the finding that the FAT-1 enzyme was found to desaturate the 18:2 of Arabidopsis membrane lipids (Spychalla et al., 1997), and by the fact that the LA product of a fungal 12 acyl-lipid fatty acid desaturase expressed in mammalian cells was exclusively located in the cellular phospholipid fraction (Kelder et al., 2001).
The principal route of triacylglyceride biosynthesis involves the transfer of acyl-CoA to a glycerol backbone via the Kennedy pathway (Kennedy, 1961), although triacylglycerols can also be synthesized from the products of phospholipid hydrolysis. Transgenic mice constitutively expressing fat-1 were found to have a significant decrease in the n-6:n-3 fatty acid ratio of milk fat (Kang et al., 2004), suggesting the movement of n-3 fatty acids from phospholipids into the triacylglycerol pool (~98% of milk fat). Interestingly, there were no apparent health problems in these transgenic mice although some may have been anticipated given the importance of phospholipid membrane composition on cellular metabolism (Hulbert, 2003). Imbalances in PUFA are attributed to many chronic disease states (Sanders, 1993), and can influence prostaglandin synthesis and fertility (Abayasekara and Wathes, 1999; Robinson et al., 2002). In vivo experiments will be required to establish what impact the coexpression of FAT-1 and FAT-2 desaturase enzymes might have on animal health and the PUFA content of milk and meat products derived from ruminant livestock.
This study demonstrates that the expression of C. elegans fatty acid desaturase genes in mammalian cells can significantly alter the cellular fatty acid profile and suggests a transgenic approach to counteract the rumen microbial biohydrogenation of unsaturated fatty acids by enabling the endogenous production of PUFA. Increasing the n-3 PUFA of beef and dairy products offers a way to improve the nutritional content of an important component of the Western diet within the realm of existing food preferences, and would provide a compelling example of how biotechnology could be employed to produce functional foods for the enhancement of human health.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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Received for publication December 8, 2004. Accepted for publication January 6, 2005.
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This article has been cited by other articles:
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  B. T. Kao, K. A. Lewis, E. J. DePeters, and A. L. Van Eenennaam Endogenous production and elevated levels of long-chain n-3 fatty acids in the milk of transgenic mice. J Dairy Sci, August 1, 2006; 89(8): 3195 - 3201. [Abstract] [Full Text] [PDF]
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