Adenoviral gene transfer of Caenorhabditis elegans n--3 fatty acid desaturase optimizes fatty acid composition in mammalian cells

Abstract

Omega--3 polyunsaturated fatty acids (PUFAs) are essential components required for normal cellular function and have been shown to exert many preventive and therapeutic actions. The amount of n--3 PUFAs is insufficient in most Western people, whereas the level of n--6 PUFAs is relatively too high, with an n--6/n--3 ratio of >18. These two classes of PUFAs are metabolically and functionally distinct and often have important opposing physiological functions; their balance is important for homeostasis and normal development. Elevating tissue concentrations of n--3 PUFAs in mammals relies on chronic dietary intake of fat rich in n--3 PUFAs, because mammalian cells lack enzymatic activities necessary either to synthesize the precursor of n--3 PUFAs or to convert n--6 to n--3 PUFAs. Here we report that adenovirus-mediated introduction of the Caenorhabditis elegans fat-1 gene encoding an n--3 fatty acid desaturase into mammalian cells can quickly and effectively elevate the cellular n--3 PUFA contents and dramatically balance the ratio of n--6/n--3 PUFAs. Heterologous expression of the fat-1 gene in rat cardiac myocytes rendered cells capable of converting various n--6 PUFAs to the corresponding n--3 PUFAs, and changed the n--6/n--3 ratio from about 15:1 to 1:1. In addition, an eicosanoid derived from n--6 PUFA (i.e., arachidonic acid) was reduced significantly in the transgenic cells. This study demonstrates an effective approach to modifying fatty acid composition of mammalian cells and also provides a basis for potential applications of this gene transfer in experimental and clinical settings.

Figure 1

Photomicrographs showing gene-transfer efficiency. Rat cardiac myocytes were infected with Ad.GFP (Left; control) or Ad.GFP.fat-1 (Right). Forty-eight hours after infection, cardiomyocytes were visualized with bright light (Upper) and at 510 nm of blue light (Lower). Coexpression of GFP demonstrates visually that the transgene is being expressed in cells in a high efficiency.

Figure 2

Ribonuclease protection assay of fat-1 transcript levels in cells infected with Ad.GFP (control) and myocytes infected with Ad.GFP.fat-1. Total RNA (10 μg) isolated from the cells was hybridized with antisense RNA probes, digested with ribonuclease, and resolved in denaturing PAGE gel. The fat-1 mRNA was visualized by autoradiography. A probe targeting the β-actin gene was used as a control.

Figure 3

Partial gas chromatograph traces showing fatty acid profiles of total cellular lipids extracted from the control cells infected with Ad.GFP and the cells infected with Ad.GFP.fat-1.

Figure 4

Enzyme immunoassay of prostaglandin E₂ levels in the control cells and the cells expressing fat-1 gene. Values are means ± SD of three experiments and expressed as a percentage of control. *P < 0.01.