Cloning, Expression, and Nutritional Regulation of the Mammalian Delta -6 Desaturase*

Hyekyung P. Cho, Manabu T. Nakamura, and Steven D. ClarkeDagger

From the Program of Nutritional Sciences and the Institute for Cellular and Molecular Biology, The University of Texas-Austin, Austin, Texas 78712

    ABSTRACT
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ABSTRACT
INTRODUCTION
REFERENCES

Arachidonic acid (20:4(n-6)) and docosahexaenoic acid (22:6(n-3)) have a variety of physiological functions that include being the major component of membrane phospholipid in brain and retina, substrates for eicosanoid production, and regulators of nuclear transcription factors. The rate-limiting step in the production of 20:4(n-6) and 22:6(n-3) is the desaturation of 18:2(n-6) and 18:3(n-3) by Delta -6 desaturase. In this report, we describe the cloning, characterization, and expression of a mammalian Delta -6 desaturase. The open reading frames for mouse and human Delta -6 desaturase each encode a 444-amino acid peptide, and the two peptides share an 87% amino acid homology. The amino acid sequence predicts that the peptide contains two membrane-spanning domains as well as a cytochrome b5-like domain that is characteristic of nonmammalian Delta -6 desaturases. Expression of the open reading frame in rat hepatocytes and Chinese hamster ovary cells instilled in these cells the ability to convert 18:2(n-6) and 18:3(n-3) to their respective products, 18:3(n-6) and 18:4(n-3). When mice were fed a diet containing 10% fat, hepatic enzymatic activity and mRNA abundance for hepatic Delta -6 desaturase in mice fed corn oil were 70 and 50% lower than in mice fed triolein. Finally, Northern analysis revealed that the brain contained an amount of Delta -6 desaturase mRNA that was several times greater than that found in other tissues including the liver, lung, heart, and skeletal muscle. The RNA abundance data indicate that prior conclusions regarding the low level of Delta -6 desaturase expression in nonhepatic tissues may need to be reevaluated.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
REFERENCES

Long chain polyunsaturated fatty acids such as 20:4(n-6) and 22:6(n-3) play pivotal roles in a number of biological functions including brain development, cognition, inflammatory responses, and hemostasis (1-4). Over 30% of the fatty acid in brain phospholipid consists of 20:4(n-6) and 22:6(n-3), and approximately 50% of the fatty acid in the retina is 22:6(n-3) (5, 6). An inadequate availability of 20:4(n-6) is associated with impaired nerve transmission, reduced eicosanoid synthesis, and impaired fetal growth (7-9). Recently, premature infants were found to have reduced cognitive development, apparently because they could not synthesize adequate quantities of 22:6(n-3) to meet the biological demands for proper retina function (1, 10). In addition to being vital components of membrane phospholipids and functioning in key steps of cell signaling, 20- and 22-carbon polyunsaturated fatty acids govern the expression of a wide array of genes, including those encoding proteins involved with lipid metabolism, thermogenesis, and cell differentiation (11-14).

The availability of 20- and 22-carbon (n-6) and (n-3) polyenoic fatty acids is greatly dependent upon the rate of desaturation of 18:2(n-6) and 18:3(n-3) by Delta -6 desaturase (15). Delta -6 Desaturase is a microsomal enzyme (15) and is thought to be a component of a three-enzyme system that includes NADH-cytochrome b5 reductase, cytochrome b5, and Delta -6 desaturase (16). The enzymatic activity for Delta -6 desaturase is reportedly low in most tissues except the liver (16). Consequently, the liver has been considered the primary site for the production of long chain polyenoic fatty acids (17, 18). Numerous dietary studies indicate that hepatic Delta -6 desaturase activity is induced by diets low in essential fatty acids and suppressed by diets rich in vegetable or marine oils (19, 20). In addition, Delta -6 desaturase activity is induced by peroxisome proliferators and by the administration of insulin to diabetic rats (21, 22). Unfortunately, defining the molecular determinants of Delta -6 desaturase activity, as well as characterizing its developmental pattern and tissue distribution, has been hampered by the fact that Delta -6 desaturase has been neither cloned nor reproducibly purified. Therefore, our objective was to clone the mammalian Delta -6 desaturase and utilize the cDNA to examine the tissue distribution and nutritional regulation of Delta -6 desaturase mRNA.

    EXPERIMENTAL PROCEDURES

Cloning of the Mouse Delta -6 Desaturase cDNA-- A murine cDNA (GenBank accession number W53753) displaying high homology to the amino acid sequence of Delta -6 desaturase from Synechocystis sp. was acquired and sequenced. Subsequently, a 23-base oligonucleotide primer (5'-CTTGGCATCGTGGGGAAGAGGTG-3') specific for the 5' end of murine cDNA W53753 was synthesized and utilized to screen a mouse adaptor-ligated liver cDNA library (Marathon-Ready cDNA; CLONTECH) by rapid amplification of cDNA ends-PCR.1 The PCR conditions consisted of an initial denaturation step of 94 °C for 1 min, followed by 5 cycles of 94 °C for 10 s and 72 °C for 4 min, 5 cycles of 94 °C for 10 s and 70 °C for 4 min, and, finally, 20 cycles of 94 °C for 10 s and 68 °C for 4 min. The resulting rapid amplification of cDNA ends-PCR product was cloned into pBluescript (Stratagene) and sequenced by the dideoxy chain termination method (23).

The nucleotide sequence of the PCR product was utilized to BLAST search the mouse EST database. Two mouse cDNAs (GenBank accession numbers AA237892 and AA250162) possessing 100% nucleotide homology with our PCR product were identified and acquired from Genome Systems. Clone AA250162 contained two possible AUG start codons, and the EST cDNA AA237892 contained an apparent stop codon. The two EST cDNAs were fused at the StyI restriction site, and the product was inserted into the cytomegalovirus promoter expression vector pcDNA3.1 (Invitrogen). Sequence analysis and prediction of amino acid sequence were performed using MacDNASIS pro (Hitachi), and a translation initiator codon was determined based on Kozak's rule (24).

Cloning of the Human Delta -6 Desaturase cDNA-- Using the nucleotide sequence of mouse liver Delta -6 desaturase, the EST human database was searched for a human homologue cDNA. The search identified a highly homologous human brain EST cDNA (GenBank accession number Z44979), which was purchased from Genome Systems and sequenced for verification. The 5' end of the human cDNA was extended by PCR using a human brain cDNA library (Marathon-Ready cDNA; CLONTECH). The forward oligonucleotide primer (5'-AGACTGGCAGCATGGGGAAG-3') was prepared using the 5' end of the mouse Delta -6 desaturase and designed to include the putative start codon. The reverse primer (5'-CATGGTGGGGAAGAGGTGGTG-3') was prepared from the sequence derived from human Z44979 cDNA.

Expression of the Mouse Delta -6 Desaturase-- Cellular expression of the mouse Delta -6 desaturase was performed in rat primary hepatocytes and CHO cells. Rat primary hepatocytes were isolated by collagenase perfusion and allowed to attach to a collagen-coated 60-mm culture plate in 3 ml of Waymouth 752 medium supplemented with 0.5% fetal bovine serum and 1 µM insulin and dexamethasone (25). After a 6-h attachment period, the cells were washed with phosphate-buffered saline and transfected with 6 µg of the mouse Delta -6 desaturase expression plasmid or the pcDNA3.1 expression vector alone using 6.6 µl of Lipofectin per µg of DNA (Life Technologies, Inc.). Transfection was conducted by adding the mixture of Lipofectin and DNA in the absence of fetal bovine serum. After the 12-h transfection period, the medium was replaced with the one containing either 200 µM albumin-bound 18:2, n-6 (molar ratio of fatty acid to albumin, 4:1) or albumin alone. CHO cells were grown in Kaighn's modification of Ham's F-12 medium supplemented with 10% fetal bovine serum in a 25-cm2 flask. At 80% confluence, the serum-containing medium was removed, and cells were washed with phosphate-buffered saline for transfection. A mixture of 2 µg of the mouse Delta -6 desaturase expression plasmid, 12 µl of LipofectAMINE, and 8 µl of Plus reagent (Life Technologies, Inc.) was added to cells without serum for 4 h. Subsequently, 10% serum was added to the transfection media for 8 h. After a total 12-h transfection period, the CHO cells were treated with either 200 µM albumin-bound 18:3(n-3), 20:3(n-6), or albumin alone. The hepatocytes and CHO cells were incubated with the treatment medium for 24 h and then used for fatty acid analysis.

Fatty Acid Extraction and Analysis-- Cellular fatty acid was extracted by saponifying fatty acids using 1 ml of 30% KOH and 1 ml of ethanol. Fatty acids from the treatment medium were also extracted and analyzed after 24 h of incubation. Heptadecanoic acid was added to the saponification mixture as an internal standard. After saponification, the nonsaponifiable lipids were removed by extraction with petroleum ether. Subsequently, the solution was acidified, and the fatty acids were extracted with petroleum ether. The extract was dried under nitrogen, and the residue was methylated using 14% boron trifluoride in methanol (Sigma). Methylated fatty acids were separated and quantified by gas chromatography using a fused silica glass capillary column (50 m × 530 µm internal diameter; Quadrex). The column temperature program was composed of an initial hold at 140 °C for 5 min, ramping at 5 °C per min to 220 °C, and a final hold at 220 °C for 7 min. The injector temperature was 250 °C, and the flame ionization detector temperature was 260 °C.

Nutritional Regulation of Delta -6 Desaturase Expression-- Male BALB/c mice were fed a high-glucose, fat-free diet for a 7-day adaptation period. After this period, the fat-free diet was supplemented with either 10% corn oil or 10% triolein (Sigma; 99% purity), and the mice (n = 4 mice/group) were fed for an additional 5 days. Liver tissues were removed, and microsomes were isolated by differential centrifugation. One g of liver was homogenized in 4 ml of homogenization buffer containing 50 mM potassium phosphate, pH 7.4, and 0.25 M sucrose. After a 10-min centrifugation of the homogenate at 10,000 × g, the resulting supernatant was spun at 100,000 × g for 60 min to isolate a microsomal pellet. After resuspending the pellet in homogenization buffer, 3 mg of microsomal protein were incubated in a 37 °C shaking water bath for 5 min with 1 ml of reaction mixture including 1.2 mM NADH, 3.6 mM ATP, 0.5 mM coenzyme A, 4.8 mM MgCl2, 72 mM phosphate buffer, pH 7.4, and 50 nmol of 1-14C-labeled 18:2(n-6). The reaction was stopped by adding saponification reagent, and fatty acids were saponified and methylated as described above. Radioactive 18:2(n-6) and 18:3(n-6) were separated by silver nitrate-impregnated thin layer chromatography. The radioactivity was quantified using an Ambis radio-imager. Delta -6 Desaturase enzyme activity is expressed as the percentage of 18:2(n-6) converted to 18:3(n-6) per mg of protein/min.

RNA Extraction and Northern Blot Analysis-- Total RNA was isolated from the liver of mice in the dietary study using the phenol-guanidinium isothiocyanate method (26). Twenty µg of total RNA were size-fractionated on a 1% formaldehyde gel and then transferred to a Zeta probe nylon membrane (Bio-Rad). The mouse Delta -6 desaturase probe was prepared by incorporating [32P]dCTP by PCR. The forward primer was 5'-GGACATAAAGAGCCTGCATG-3', and the reverse primer was 5'-ACTGGAAGTACATAGGGATG-3'. The Northern membrane of human tissues was purchased from Invitrogen. The radiolabeled probe for the human tissue blot was a 200-base pair PCR fragment of human Delta -6 desaturase using primers of 5'-GGCAAGAACTCAAAGATCAC-3' and 5'-GAGAGGTAGCAAGAACAAAG-3'. The autoradiographic signal was quantified using Instant Imager (Packard).

    RESULTS

Cloning Mouse and Human Delta -6 Desaturase-- The mouse EST database was searched for mammalian homologues using the amino acid sequence for Delta -6 desaturase from the photosynthetic cyanobacterium Synechocystis sp. (27). A mouse cDNA that had a 60% similarity to a 46-amino acid sequence of Synechocystis Delta -6 desaturase was identified. A 1508-base pair cDNA sequence for mouse liver Delta -6 desaturase was acquired using a combination of ligation-mediated PCR screening of a mouse liver cDNA library and BLAST searches of the mouse EST database (Fig. 1A). Sequence analysis revealed the presence of two in-frame methionine codons located at positions 75 and 126. In addition, a TGA termination sequence was identified at position 1407. Kozak's rule, which predicts that the favored eukaryotic translation initiation sequence resides in the sequence of AXXATGG (24), indicated that the first of the two ATG codons was the preferred initiation codon for the putative Delta -6 desaturase. The apparent ORF between the first ATG codon and the TGA termination codon predicted a peptide consisting of 444 amino acids and having a size of 52.2 kDa. The human brain cDNA homologue for Delta -6 desaturase contains an initiation codon and a termination codon that are perfectly aligned with the initiation and termination codons of the mouse cDNA (Fig. 1A). Moreover, the amino acid sequence derived from the ORF revealed that 87% of the amino acid sequence for the mouse and human homologues was identical, and 96% of the sequence had similarity (Fig. 1B). A search of the Swiss Protein Database indicated that the putative Delta -6 desaturase sequence was unique and shared very little amino acid homology with any other mammalian proteins including the murine stearoyl-CoA desaturase (Delta -9 desaturase) (28).


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Fig. 1.   Alignment of the predicted amino acid sequences for mouse and human Delta -6 desaturase. A, schematic diagram of the ORF and untranslated regions for mouse and human Delta -6 desaturase. The hatched box indicates an ORF of 1332 nucleotides, and the open boxes represent untranslated regions. The human cDNA contains a polyadenylation signal AATAAA at the 3' end. B, a comparison of the amino acid sequences for mouse and human Delta -6 desaturase predicted by the nucleotide sequence of the ORFs. Both mouse and human ORFs encode 444 amino acids. Identical amino acids are paired by vertical lines, and conserved amino acids are matched by colons. The cytochrome b5-like domain is underlined. Transmembrane domains are shown in shaded areas, and three histidine-rich domains are in bold.

Structural Characteristics of Mammalian Delta -6 Desaturase-- The enzymatic activity of mammalian Delta -6 desaturase is associated with the microsomal membrane fraction (15). Consistent with such membrane involvement, the predicted amino acid sequence for Delta -6 desaturase indicated that the peptide contains 52% nonpolar amino acids, and a hydropathy profile revealed the presence of two membrane-spanning domains that are characteristic of membrane-anchored proteins (Figs. 1B and 2). In addition, the amino terminus of the Delta -6 desaturase peptide contains a hydrophilic domain of 54 amino acids that is highly homologous with the heme-binding domain of cytochrome b5 (Fig. 3A). This cytochrome b5-like domain is also found in the Delta -6 desaturases from Borago officinalis (29) and Caenorhabditis elegans (30) (Fig. 3A). The His53 and His76 residues located within this domain of the mammalian Delta -6 desaturase are exactly aligned with the two heme-binding histidines in cytochrome b5 (31). Moreover, these two histidines are surrounded by charged amino acids that may contribute to the stabilization of the heme-histidine complex (31). In addition, the sequence 53HPGG56 predicts the existence of a dramatic beta -turn that may render His53 more accessible to heme iron binding (31).


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Fig. 2.   Hydropathy profile of mouse (A) and human (B) Delta -6 desaturase. The hydropathic pattern for Delta -6 desaturase was plotted using the method of Kyte-Doolittle, and the amino acid sequences were predicted by the respective ORFs. Bars,the transmembrane regions. Boxed H, locations of histidine-rich regions.


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Fig. 3.   A comparison of the cytochrome b5-like and histidine-rich domains for mammalian and nonmammalian Delta -6 desaturases. A, a comparison of the cytochrome b5-like domain for mammalian and nonmammalian Delta -6 desaturase (D6D). A comparison of the amino acid sequence within the cytochrome b5-like domain of mouse, human, plant (B. officinalis; Ref. 29), and C. elegans (30) Delta -6 desaturases reveals a high level of homology with a comparable domain within mammalian cytochrome b5 (Cyto.b5) (31). Amino acids that are identical between the Delta -6 desaturases and cytochrome b5 are highlighted in black; amino acids that are highly homologous between the desaturases and cytochrome b5 are highlighted in gray. Asterisk, two heme-binding histidines found in cytochrome b5 (31). B, the three histidine-rich regions conserved in membrane desaturases. The histidines within these regions are highlighted in black. The amino acids that are identical in all the Delta -6 desaturases listed are highlighted in dark gray.

A second noteworthy feature of the mammalian Delta -6 desaturase is the presence of three histidine-rich regions (Fig. 3B). Regions I (HX3H) and II (HX2HH) are located between the two transmembrane domains, and region III (HH) is located near the carboxyl terminus of the peptide. These histidine-rich regions are also found in plant membrane desaturases and mammalian stearoyl-CoA desaturase and reportedly bind non-heme iron that is required for enzymatic activity (32).

Expression of Delta -6 Desaturase-- The predicted structural characteristics of the mouse and human peptides strongly suggested that the cDNAs did in fact correspond to mammalian Delta -6 desaturase. To confirm this conclusion, the ORF for the mouse Delta -6 desaturase was expressed in primary cultures of rat hepatocytes and in CHO cells. Fatty acid analysis revealed that hepatocytes transfected with the vector containing the Delta -6 desaturase ORF were capable of synthesizing the Delta -6 desaturase product 18:3(n-6) from 18:2(n-6) (Fig. 4A). On the other hand, hepatocytes transfected with vector alone produced no detectable 18:3(n-6) product (Fig. 4B). Similarly, CHO cells expressing Delta -6 desaturase readily converted the Delta -6 desaturase substrate 18:3(n-3) to the Delta -6 desaturase product 18:4(n-3), whereas nontransfected CHO cells were unable to produce detectable levels of 18:4(n-3) (Fig. 4, C and D). In contrast, providing CHO cells with the Delta -5 desaturase substrate 20:3(n-6) did not lead to the production of the Delta -5 desaturase product 20:4(n-6) (data not shown). These data conclusively demonstrate that the mouse and human ORFs do in fact encode mammalian Delta -6 desaturase.


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Fig. 4.   Expression of mouse Delta -6 desaturase in rat hepatocytes and CHO cells. A shows the conversion of 18:2(n-6) to 18:3(n-6) by hepatocytes that were transfected with the pcDNA3.1 vector containing the mouse Delta -6 desaturase (D6D) ORF. When hepatocytes were transfected with pcDNA3.1 vector lacking the Delta -6 desaturase ORF, there was no detectable conversion of 18:2(n-6) to 18:3(n-6) (B). The media of CHO cells incubated with albumin-bound 18:3(n-3) and transfected with the Delta -6 desaturase expression vector pcDNA3.1 contained 18:4(n-3) (C), whereas the media of nontransfected CHO cells contained no detectable 18:4(n-3) (D). Retention times for the fatty acids are shown above the respective peaks. The identity of each peak was confirmed using individual fatty acid methyl ester standards.

Nutritional Regulation of Delta -6 Desaturase Expression-- The enzymatic activity of Delta -6 desaturase increases when animals are fed an essential fatty acid-deficient diet, whereas it decreases when polyunsaturated fatty acids are ingested (16, 19, 20). Using the mouse cDNA for Delta -6 desaturase, we have found that the suppression of hepatic Delta -6 desaturase enzymatic activity associated with the ingestion of polyunsaturated fat (i.e. corn oil) is paralleled by a comparable reduction in Delta -6 desaturase mRNA abundance (Fig. 5, A and B). Interestingly, whereas the dominant transcript of hepatic Delta -6 desaturase is approximately 4.0 kb in size, the mouse liver also contains a minor transcript that is approximately 2.2 kb (Fig. 5B). Both transcripts appeared to be suppressed by dietary corn oil to the same degree. In addition, hybridizing the Northern blot with sequences from the 5', middle, and 3' regions of the Delta -6 desaturase ORF yielded the same outcomes with respect to the abundance and dietary response of the 2.2-kb transcript (data not shown). The reason for these two different transcripts remains unknown.


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Fig. 5.   Nutritional regulation and tissue distribution of mammalian Delta -6 desaturase. Mice were fed a high-glucose diet containing 10% corn oil or 10% triolein. Hepatic Delta -6 desaturase activity, which is expressed in A as means ± S.E., was significantly lower in mice fed corn oil than in mice fed triolein (p < 0.001). The abundance of hepatic Delta -6 desaturase mRNA was determined by Northern analysis (B). The abundance of the 4.0- and 2.2-kb Delta -6 desaturase transcripts was quantified by radio-imaging. The cpm of the 32P-labeled probe associated with the 4.0- and 2.2-kb transcripts was 2509 ± 154 and 327 ± 17; it was 1264 ± 66 and 185 ± 9 for the triolein and corn oil groups, respectively (p < 0.003). C depicts the abundance of Delta -6 desaturase mRNA found in a variety of adult male human tissues. Each lane contains 20 µg of total RNA. Unlike mice, only one Delta -6 desaturase transcript with an approximate size of 3.2 kb was detected in human tissues. Comparable results were obtained with three different Northern blots.

Delta -6 Desaturase mRNA Distribution in Human Tissues-- Northern analysis of Delta -6 desaturase expression revealed that human Delta -6 desaturase mRNA is a single transcript of approximately 3.2 kb and is expressed in a wide array of tissues including the brain, liver, lung, and heart (Fig. 5C). The level of Delta -6 desaturase mRNA in the liver was approximately the same as that found in the lung and heart, but the abundance of Delta -6 desaturase in the human brain was severalfold higher (Fig. 5C). In addition to the tissues examined by Northern analysis, a search of the EST database revealed that Delta -6 desaturase mRNA is expressed in the human fetus and fetal heart as well as in the 13-day-old mouse embryo heart.

    DISCUSSION

The purification and characterization of mammalian Delta -6 desaturase have been difficult because of its instability. In fact, there has been only one report, in 1981, that describes the purification of a putative linoleoyl-CoA desaturase from rat liver (33). Because of the problems encountered in the purification of Delta -6 desaturase, we have used the EST database to clone and characterize the mouse and human Delta -6 desaturase enzyme. Interestingly, a comparison of the rat liver linoleoyl-CoA desaturase with the Delta -6 desaturase peptide predicted by the ORF of both the mouse and human cDNAs indicates that the two proteins are markedly different. First, the ORF for mouse and human Delta -6 desaturase predicts a protein that is 52.2 kDa, whereas the size of the linoleoyl-CoA desaturase was cited to be 66 kDa (33). Second, the nucleotide sequence of the mouse and human Delta -6 desaturase ORFs predicts that these peptides contain 30 histidines (Fig. 1B). Moreover, many of these histidines are organized into distinct histidine-rich domains. Such domains are characteristic of all membrane-associated desaturases (32). In contrast, the reported amino acid composition of linoleoyl-CoA desaturase indicates that it contains only 15 histidine residues (33). Unfortunately, sequence information for linoleoyl-CoA desaturase is not available, because the purification of linoleoyl-CoA desaturase has never been replicated since the initial report. Clearly, the Delta -6 desaturase and the putative linoleoyl-CoA desaturase are distinctly different proteins. It is possible the liver contains two Delta -6 desaturase enzymes. In fact, metabolic studies suggest that there may be two isoforms of Delta -6 desaturase (34, 35): (a) one that catalyzes the initial desaturation of 18:2(n-6) or 18:3(n-3), and (b) another that catalyzes the conversion of 24:5(n-3) to 24:6(n-3). The cloning of the Delta -6 desaturase should now permit us to determine whether isoforms of Delta -6 desaturase do exist.

In addition to the histidine-rich domains, the mammalian Delta -6 desaturase contains a distinct cytochrome b5-like domain that is also characteristic of plant (Borage) and C. elegans Delta -6 desaturases (29, 30) but is not a component of the mammalian Delta -9 desaturase (36). Early reconstitution studies with Delta -9 desaturase indicated that the conversion of 18:0(n-9) to 18:1(n-9) required Delta -9 desaturase, cytochrome b5 reductase, and cytochrome b5 itself (36). It has been assumed from these early studies that all mammalian desaturases require cytochrome b5 for enzymatic activity (16, 36). However, the cytochrome b5-like domain of yeast OLE1 was recently reported to replace the requirement for cytochrome b5; i.e. desaturation occurred in the absence of cytochrome b5, and removal of the cytochrome b5-like domain rendered the OLE1 enzyme inactive (37). This observation raises the possibility that cytochrome b5 reductase transfers electrons to the catalytic domain of the Delta -6 desaturase via the cytochrome b5-like domain, and not via cytochrome b5 per se.

Hepatic Delta -6 desaturase enzymatic activity varies with hormonal and nutritional manipulation (15, 16, 20, 38). For example, insulin deficiency and fasting reduce Delta -6 desaturase enzymatic activity, whereas the administration of insulin or refeeding increases its activity (39). In addition to being affected by fasting and feeding, hepatic Delta -6 desaturase enzymatic activity is highly dependent upon the composition of dietary fat (16). Specifically, the ingestion of fats that are low in essential fatty acids (e.g. butter) results in higher levels of enzyme activity than the consumption of fats (e.g. corn oil) that are rich in essential fatty acids (16). Northern analysis indicates that the increase in hepatic Delta -6 desaturase activity associated with the consumption of an essential fatty acid-deficient diet is paralleled by a comparable increase in the hepatic abundance of Delta -6 desaturase mRNA (Fig. 5). Thus, it appears that the activity of hepatic Delta -6 desaturase is largely regulated by pretranslational events. However, this may not be the case in all tissues. Specifically, Delta -6 desaturase activity is reportedly very low in nonhepatic tissues (16-18). Because of this low enzymatic activity in nonhepatic tissues, the liver has been considered to be the primary site of 20:4(n-6), 20:5(n-3), and 22:6(n-3) production for peripheral tissue utilization (17). However, Northern analysis of RNA from a number of different human tissues challenges this concept (Fig. 5C). For example, the level of Delta -6 desaturase mRNA in the human liver was comparable to that found in the human lung and heart. Moreover, the abundance of Delta -6 desaturase mRNA in the adult human brain was severalfold greater than that in the human liver (Fig. 5C). This high level of expression is certainly very consistent with the fact that >30% of the human brain phospholipid consists of 20- and 22-carbon polyenoic fatty acids (5, 6, 40). However, such high expression is in conflict with the reports that brain microsomes have a rate of Delta -6 desaturation that is only 10-15% of that found in the liver (18, 41). These data suggest that Delta -6 desaturase enzymatic activity may be determined by tissue-specific mechanisms that involve both pre- and post-translational events.

In conclusion, Delta -6 desaturase catalyzes the rate-limiting step in the conversion of 18:2(n-6) and 18:3(n-3) to the long chain polyenoic fatty acids 20:4(n-6) and 20:5(n-3) and 22:6(n-3), respectively (15). These long chain polyenoic fatty acids are essential for a large number of biological functions including inflammatory responses (4), brain development (2), retina function and cognition (1, 3), signal transduction (42, 43), reproduction (4), fetal growth (9), cell differentiation (14), and gene regulation (11-13). Not surprisingly, physiological conditions that are associated with low levels of Delta -6 desaturase activity may have a pronounced impact on a wide array of biological functions. For example, an impaired conversion of 18:2(n-6) to 18:3(n-6) appears to be associated with reduced nerve conductivity in human diabetics (7). Similarly, the low rate of 18:3(n-3) conversion to 20:5(n-3) and 22:6(n-3) observed in newborn infants is highly correlated with impaired retina function and reduced cognitive development (1). Now that the Delta -6 desaturase has been cloned, we can begin to define the role that Delta -6 desaturation may play in an apparently wide array of physiological processes.

    FOOTNOTES

* This work was supported by the National Institutes of Health Grant DK 52573 and the sponsors of the M. M. Love Chair in Nutritional, Cellular and Molecular Sciences at the University of Texas-Austin (S. D. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: 115 GEA, The University of Texas at Austin, Austin, TX 78712. Tel.: 512-232-1537; Fax: 512-471-5630; E-mail: stevedclarke{at}mail.utexas.edu.

    ABBREVIATIONS

The abbreviations used are: PCR, polymerase chain reaction; ORF, open reading frame; EST, expressed sequence tag; CHO, Chinese hamster ovary; kb, kilobase pair(s)..

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ABSTRACT
INTRODUCTION
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